Morphometrical Study of Polysialylated Neural Cell Adhesion Molecule Positive Cells in Rat Pups Hippocampus Following Induction of Seizure during Pregnancy

ABSTRACT Background:The polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed in developing brain. Fetal brain damage is caused by different conditions such as seizure and hypoxia. The present study was designed to investigate the effect of maternal seizures on the number of PSA-NCAM positive cells in pup's hippocampus. Methods: Female Wistar rats were divided into four groups: (a) kindled rats which received PTZ (40 mg/kg, i.p.) during pregnancy from embryonic day 14-19 (E14-E19) every 48 h, (b) kindled rats which did not receive PTZ during pregnancy, (c) non-kindle, pregnant rats which received PTZ injection (40 mg/kg, i.p.) during pregnancy from E14 to E19 every 48 h, and (d) non-kindle, pregnant rats which received injection with an equal volume of normal saline as sham controls. At postnatal day 14 (PD 14 ), rat pups were perfused, and their brain were fixed, embedded and coronal sections stained by immunohistochemistry method. The number of PSA-NCAM positive cells per unit area in the pup's hippocampus was counted. Results: The number of PSA-NCAM positive cells in the CA1, CA3, and DG fields of pup's hippocampus, which was obtained from mothers who experienced PTZ injection during pregnancy, was decreased approximately 2.6 (P = 0.001), 2 (P = 0.001), and 2.1 (P = 0.001) times compared with non-PTZ treated maternal groups, respectively. Conclusions: Our study showed that maternal seizures reduced the number of neurons and also PSA-NCAM positive cells per unit area in the offspring hippocampus that it may cause impairment in hippocampal functions. Iran. Biomed. J. 15 (4): 157-163, 201

Effects of Hypothyroidism on Proliferation and Programmed Cell-Death in Rat Ovarian Granulosa Cells

Hypothyroidism is the most common endocrine disease, after diabetes. Thyroid hormones are essential for genital organs function. In this study, we aimed to determine the apoptotic and cell proliferation indexes resulting from reduced thyroid hormones in rat ovarian follicles. For the purpose of this study, 20 female mature Wistar rats were divided into test and control groups. The test group underwent chemical thyroidectomy by receiving 500 mg/l propylthiouracil added to drinking water. The control group only received ordinary drinking water. After three weeks, the rats were sacrificed and their ovaries were removed and fixed for tissue preparation. Triphosphate-biotin nick end-labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunohistochemical techniques were applied to determine apoptosis and cell proliferation variations. Our findings revealed that apoptotic index significantly diminished in large antral follicles. There were no significant differences between the two groups in terms of primary and pre-antral follicles. No TUNEL-positive cell was noted in primordial follicles in the both groups. Cell proliferation index revealed a significant decrease in follicular growth of pre-antral to graafian follicles in the hypothyroid group. PCNA-positive cells were not observed in primordial follicles in the both groups. The results of the study suggested that reduced thyroid hormones lead to a wide range of hormonal changes, and factors existing in follicular fluid, especially in large antral follicles, undergo transformations that affect apoptotic and cell proliferation indexes. The process of follicular growth occurs by entrance of follicles to the next growth phase without inducing sufficient potentiality, and the produced ovules might be healthy or morphologically defected

Effects of Hypothyroidism on Proliferation and Programmed Cell-Death in Rat Ovarian Granulosa Cells

Hypothyroidism is the most common endocrine disease, after diabetes. Thyroid hormones are essential for genital organs function. In this study, we aimed to determine the apoptotic and cell proliferation indexes resulting from reduced thyroid hormones in rat ovarian follicles. For the purpose of this study, 20 female mature Wistar rats were divided into test and control groups. The test group underwent chemical thyroidectomy by receiving 500 mg/l propylthiouracil added to drinking water. The control group only received ordinary drinking water. After three weeks, the rats were sacrificed and their ovaries were removed and fixed for tissue preparation. Triphosphate-biotin nick end-labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunohistochemical techniques were applied to determine apoptosis and cell proliferation variations. Our findings revealed that apoptotic index significantly diminished in large antral follicles. There were no significant differences between the two groups in terms of primary and pre-antral follicles. No TUNEL-positive cell was noted in primordial follicles in the both groups. Cell proliferation index revealed a significant decrease in follicular growth of pre-antral to graafian follicles in the hypothyroid group. PCNA-positive cells were not observed in primordial follicles in the both groups. The results of the study suggested that reduced thyroid hormones lead to a wide range of hormonal changes, and factors existing in follicular fluid, especially in large antral follicles, undergo transformations that affect apoptotic and cell proliferation indexes. The process of follicular growth occurs by entrance of follicles to the next growth phase without inducing sufficient potentiality, and the produced ovules might be healthy or morphologically defected

The effects of nano-silver and garlic administration during pregnancy on neuron apoptosis in rat offspring hippocampus

Objective(s):The aim of this study was to investigate the effects of nano-silver and garlic administration during pregnancy on neuron apoptosis in rat offspring hippocampus. Materials and Methods: Fifty pregnant wistar rats were randomly divided into five groups: 1- nano- silver (N.S) group; 30 mg/kg of N.S treated via gavage. 2- Control (C) group, administrated with distilled water via gavage. 3- N.S and garlic (N.S+G) group; N.S (30 mg/kg) and garlic juice (1 ml/100 g) treated via gavage simultaneously. 4- Garlic group (G); garlic juice (1 ml/100 g) administrated via gavage, 5- normal (N) without any intervention. All the interventions were done during pregnancy (21 days). Finally, the brains of rat offspring were removed to use for nano-silver level measurement and TUNEL staining. The mean of TUNEL positive cell numbers per unit area (NA) in different regions of hippocampus were compared in all animal groups. Results: The results revealed a significant increase of hippocampus nano-silver level in N.S and N.S+G groups comparing to N group (

Lectin Histochemical Study of Vasculogenesis During Rat Pituitary Morphogenesis

Objective(s) The aim of this study was to investigate glycoconjugates distribution patterns as well as their changes during the course of pituitary portal vasculogenesis and angiogenesis.Materials and MethodsFormalin fixed paraffin sections of 10 to 20 days of Sprague Dawly rat fetuses were processed for histochemical studies using four different horseradish peroxidase (HRP) conjugated lectins. Orange peel fungus (OFA), Vicica villosa (VVA), Glycine max (SBA) and Wistaria floribunda (WFA) specific for α-L-Fucose, D-Gal, α, ß-D-GalNAc and D- GalNAc terminal sugars of glycoconjugates respectively.ResultsOur finding indicated that adenohypophysal cells reacted with OFA on gestational day 10 (E10) and increased progressively to E14. Staining intensity did not change from days 14 to17, then after increased following days to E20 significantly (P< 0.05). A few cells around Rathke’s pouch reacted with VVA on E13, increased to E14 and decreased significantly afterward (P< 0.05). Reaction of some cells around Rathke’s pouch reacted with SBA on E14. This visible reaction was the same as E18 and decreased later (P< 0.05). Many cells around Rathke’s pouch reacted with WFA on E13 and increased on E 14 and E15 and decreased thereafter (P< 0.05).ConclusionReactions of OFA and other tested lectins with endothelial cells around Rathke’s pouch and developing pars distalis were different. These results suggest that embryonic origin of hypophiseal pituitary portal (HPP) system endothelial cells are not the same and our finding also indicated that glycoconjugates with terminal sugars α-L-Fucose, D-Gal, α, ß-D-GalNAc may play critical role(s) in cell interactions and tissue differentiations such as vasculogensis and angiogenesis as well as other developmental precursors in formation of the pituitary gland

Regulatory Changes of N-Acetylgalactosamine Terminal Sugar in Early Mouse Embryonic Paraxial Mesenchyme

Objective: The development of vertebrae is a complex phenomenon that is correlated with distinct morphological and biochemical alterations in the paraxial mesenchyme and glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique.Materials and Methods: In this descriptive-analytic study, B4G fixed paraffin sections of 9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven different HRP-labelled lectins: Glycin max (SBA), Maclura pomifera (MPA), Wistaria floribunda (WFA), Vicia villosa (VVA) which all of them are specific for N-acetylgalactosamine (GalNAc), Ulex europius (UEA1, binds to α-L-fucose), wheat germ agglutinin (WGA, binds to sialic acid), and Griffonia simplicifolia (GSA1-B4, binds to galactose terminal sugars). The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins’ reactions with the specimen, from negative (-) to severe (+++). Data was analysed with SPSS software (version 11.5) and the non-parametric Kruskal Wallis test; p<0.05 was considered significant.Results: Our findings showed that among the tested lectins, only GalNAc residue sensitive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continued to GD15 constantly, while MPA reactions continued strongly to GD12, significantly decreased thereafter (p<0.001), and then disappeared. VVA and SBA bindings initiated weakly on GD10 and continued to GD12 without changing. These reactions increased significantly (p<0.001) thereafter, became severe to GD14, and later disappeared. The other tested lectins did not reveal regulated changes.Conclusion: According to these findings it can be concluded that only the GalNAc terminal sugar showed temporally regulated changes during the early embryonic development of vertebrae in mice. Therefore it most likely plays a key role (s) in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other tested terminal sugars may have no role in this phenomenon

Immunohistochemical study of type III collagen expression during pre and post-natal rat skin morphogenesis

Objective(s):Skin extracellular matrix, which contains type I and type III collagens, is involved in skin development. The aim of this study was to investigate type III collagen distribution pattern as well as its changes during pre and post-natal skin morphogenesis in rats. Materials and Methods: Ventral skins of Wistar rat embryos at different stages from 10 to 20 gestational day (E10-E20) and also one month and one year post natal rat pups were fixed in normalin, embedded in paraffin and 5 µm thick sections were incubated with Anti type III collagen antibody. In order to detect staining intensity, the reactions were observed and graded by three examiners separately. Kruskal-Wallis non-parametric statistical test and SPSS software version 11.5 were used to compare differences between samples. Results: Immunoreactivity of type III collagen was distributed weakly in the mesenchymal connective tissue on day 10 (E10). The observed reaction was increased onE12 and E14. This reaction was clear in basement membrane, relatively intensive in dermal papillae and moderate in dermal reticularis on E14. This immunoreactivity pattern was increased afterward on E16, not changed on E18 and decreased in dermal reticularis on E20. The density of collagen type III in dermal papillae and dermal reticularis in skin of one year old rats were decreased comparing to one month old rats. Conclusion: Our results showed that type III collagen is expressed and timely regulated during pre and post natal rat skin morphogenesis

Role of the Nitrergic System of the Cuneiform Nucleus in Cardiovascular Responses in Urethane-Anesthetized Male Rats

Background: The presence of nitric oxide (NO) in the cuneiform nucleus (CnF) has been previously shown. In this study, NG-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of NO synthase), L-arginine (L-Arg) (a precursor of NO), and sodium nitroprusside (SNP) (a donor of NO) were microinjected into the CnF and cardiovascular responses were investigated. Methods: Seventy male rats were divided into 7 groups (n=10 each): 1) saline, 2 and 3) L-NAME (30 and 90 nmol), 4 and 5) L-Arg (20 and 60 nmol), and 6 and 7) SNP (9 and 27 nmol). After anesthesia, the femoral artery was cannulated and cardiovascular parameters were recorded using a PowerLab system. Time course changes in mean arterial pressure (ΔMAP) and heart rate (ΔHR) were calculated and compared with those in the control group (repeated measures ANOVA). Maximum ∆MAP and ∆HR were also compared with those in the control group (independent sample t test). Results: ∆MAP with both doses of L-NAME (30: P=0.026 and 90: P=0.007) and ∆HR with the higher dose (P=0.034) were significantly higher than those in the control group. Maximal ∆MAP with both doses (P<0.01 and P<0.001, n=10) and maximal ∆HR with the higher dose (P<0.01) were significantly higher than those in the control group. Changes in L-Arg with both doses were not significantly higher than those in the control group (P=0.26, n=8). ∆MAP and ∆HR of SNP only with the higher dose were significantly lower than those in the control group (P=0.006 and P=0.035), and maximal responses with the higher dose were lower than those in the control group (∆MAP: P<0.01 and ∆HR: P<0.05, n=7). Conclusion: Our results showed that the nitrergic system of the CnF had an inhibitory effect on central cardiovascular regulation

Zinc- and Copper-Doped Mesoporous Borate Bioactive Glasses: Promising Additives for Potential Use in Skin Wound Healing Applications

In this study, zinc (Zn)- and copper (Cu)-doped 13-93B3 borate mesoporous bioactive glasses (MBGs) were successfully synthesized using nitrate precursors in the presence of Pluronic P123. We benefited from computational approaches for predicting and confirming the experimental findings. The changes in the dynamic surface tension (SFT) of simulated body fluid (SBF) were investigated using the Du Noüy ring method to shed light on the mineralization process of hydroxyapatite (HAp) on the glass surface. The obtained MBGs were in a glassy state before incubation in SBF. The formation of an apatite-like layer on the SBF-incubated borate glasses was investigated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The incorporation of Zn and Cu into the basic composition of 13-93B3 glass led to changes in the glass transition temperature (Tg) (773 to 556 °C), particle size (373 to 64 nm), zeta potential (−12 to −26 mV), and specific surface area (SBET) (54 to 123 m2/g). Based on the K-means algorithm and chi-square automatic interaction detection (CHAID) tree, we found that the SFT of SBF is an important factor for the prediction and confirmation of the HAp mineralization process on the glasses. Furthermore, we proposed a simple calculation, based on SFT variation, to quantify the bioactivity of MBGs. The doped and dopant-free borate MBGs could enhance the proliferation of mouse fibroblast L929 cells at a concentration of 0.5 mg/mL. These glasses also induced very low hemolysis (Pseudomonas aeruginosa. In summary, we showed that Cu-/Zn-doped borate MBGs can be fabricated using a cost-effective method and also show promise for wound healing/skin tissue engineering applications, as especially supported by the cell test with fibroblasts, good compatibility with blood, and antibacterial properties