Maternal N-Acetyl-Cysteine Prevents Neonatal Hypoxia-Induced Brain Injury in a Rat Model

Perinatal hypoxia is a major cause of infant brain damage, lifelong neurological disability, and infant mortality. N-Acetyl-Cysteine (NAC) is a powerful antioxidant that acts directly as a scavenger of free radicals. We hypothesized that maternal-antenatal and offspring-postnatal NAC can protect offspring brains from hypoxic brain damage.Sixty six newborn rats were randomized into four study groups. Group 1: Control (CON) received no hypoxic intervention. Group 2: Hypoxia (HYP)-received hypoxia protocol. Group 3: Hypoxia-NAC (HYP-NAC). received hypoxia protocol and treated with NAC following each hypoxia episode. Group 4: NAC Hypoxia (NAC-HYP) treated with NAC during pregnancy, pups subject to hypoxia protocol. Each group was evaluated for: neurological function (Righting reflex), serum proinflammatory IL-6 protein levels (ELISA), brain protein levels: NF-κB p65, neuronal nitric oxide synthase (nNOS), TNF-α, and IL-6 (Western blot) and neuronal apoptosis (histology evaluation with TUNEL stain). Hypoxia significantly increased pups brain protein levels compared to controls. NAC administration to dams or offspring demonstrated lower brain NF-κB p65, nNOS, TNF-α and IL-6 protein levels compared to hypoxia alone. Hypoxia significantly increased brain apoptosis as evidenced by higher grade of brain TUNEL reaction. NAC administration to dams or offspring significantly reduce this effect. Hypoxia induced acute sensorimotor dysfunction. NAC treatment to dams significantly attenuated hypoxia-induced acute sensorimotor dysfunction. Prophylactic NAC treatment of dams during pregnancy confers long-term protection to offspring with hypoxia associated brain injury, measured by several pathways of injury and correlated markers with pathology and behavior. This implies we may consider prophylactic NAC treatment for patients at risk for hypoxia during labor.

Genistein attenuated gastric inflammation and apoptosis in Helicobacter pylori-induced gastropathy in rats

Background Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis, peptic ulcer diseases and cancer. Genistein (4′,5,7-trihydroxyisoflavone), a tyrosine-specific-protein kinase inhibitor, has been shown to exert an anti-inflammatory property. The aim of this study was to examine the treatment effects of genistein and its mechanisms in rats with H. pylori infection. Methods Eighteen male Sprague-Dawley rats were divided into three groups (6 rats per group): (1) control group (Con); (2) H. pylori infected group (HP): the rats were inoculated with H. pylori (108− 1010 CFU/mL; 1 mL/rat.) for 3 consecutive days; and (3) HP + genistein group (HP + Gen): the rats were inoculated with H. pylori as above. Then, they were gavaged with genistein (16 mg/kg BW) for 14 days. Gastric tissue was used for the determination of nuclear factor (NF)-κB expression by immunohistochemistry (IHC), degree of apoptosis by the terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) reaction, and histopathology. Serum samples were used to measure the levels of tumor necrosis factor-alpha (TNF-α) and cytokine-induced neutrophil chemoattractant-1 (CINC-1). Results Rats in the HP group had significantly higher levels of pro-inflammatory mediators, NF-κB expression and apoptotic cells when compared with the Con group, and these markers significantly decreased in HP + Gen group when compared with the HP group. The histopathology of HP group showed moderate gastric inflammation and many HP colonization. Gastric pathology in HP + Gen group demonstrated the attenuation of inflammatory cell infiltration and H. pylori colonization. Conclusion Genistein exerted its gastroprotective effects through the reduction of pro-inflammatory mediators, nuclear receptor NF-κB expression and gastric mucosal apoptosis in rats with H. pylori-induced gastropathy.

Sambucus Ebulus Fruits Extract Ameliorates Eosinophilic Nasal Polyposis: An in vitro Study

Background: The Sambucus Ebulus (ES) L. has an anti-inflammatory role by reducing the expression of pro-inflammatory genes and free radicals. The aim of this study was to evaluate the anti-inflammatory effect of hydroalcoholic extract of SE fruits on eosinophilic nasal polyposis (NP) in Vitro.Method: The chemical composition of SE fruits extract was determined by gas chromatography-mass spectrometry. Polyp Samples were collected from 40 patients and were cut into four pieces (control and case groups). Tissue fragments were treated with 0 (control), 50, 315 and 1000µg/ml of SE FRUIT extract in a culture medium for 24 hours. Apoptosis was detected by TUNEL reaction reagent. Real time-PCR was employed to evaluate the expression of Bax and Bad genes. ELISA was used for assay of IL-5 and GM-CSF.Results: Our findings showed SE fruits extract increased apoptosis in nasal polyp tissues compared to the control group (P<0.001). Also, GM-CSF level in the experimental groups was significantly lower than that in the control group (P<0.001). In contrast, the level of IL-5 was not significantly different in the treatment and control groups (P>0.05). Bax and Bad expression showed a significant increase in the experimental groups compared to the control group (P<0.001).Conclusion: Our findings showed that SE fruits hydroalcoholic extract ameliorates NP trough elevation of apoptosis and reduction of inflammatory markers. Our experimental data revealed that SE fruits extract plays an important role in the treatment of nasal polyp through inducing apoptosis and reducing the survival of inflammatory cells.

Thermal stress induces heat shock protein 70 and apoptosis during embryo development in a Neotropical freshwater fish

Fish embryos are particularly vulnerable to temperature changes, with the effects varying with developmental stage. The major aim of the present study was to analyse the relationship between apoptosis and heat shock protein (HSP) 70 during embryo development under thermal stress conditions. To this end, Prochilodus lineatus embryos at the blastopore closure stage were subjected to one of three thermal treatments for 1h (Group 1, 25°C (control); Group 2, 20°C; Group 3, 30°C) and then examined at 0, 4 and 8h posttreatment (h.p.t.). The viability of embryos was highest in Group 1 (81.33±16.65%), followed by Group 3 and Group 2 (75.33±12.10% and 68.67±16.86% respectively), with significant difference between Groups 1 and 2 (P&lt;0.05). At 0h.p.t., embryos subjected to thermal stress (Group 3) had a significantly higher number of terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL)- and caspase-3-labelled cells, and a lower number of HSP70-positive cells than those in the control group. At 4h.p.t., there was a decrease in the TUNEL reaction and an increase in HSP70 in embryos in Group 3. At 8h.p.t., the size of Group 3 embryos was significantly smaller than that of Group 1 embryos. The results indicate a cytoprotective role for HSP70, regulating caspase-3-mediated apoptosis during embryo development of P. lineatus; however, this mechanism is not effective in controlling embryo viability and larval malformations.

An Investigation of the Effects of Curcumin on the Changes in the Central Nervous System of Rats Exposed to Aroclor 1254 in the Prenatal Period

Background & Objective: Aroclor 1254 is a widespread toxic compound of Polychlorinated Biphenyls (PCBs), which can create significant nervous problems. No remedies have been found to date. The aim of this study was to reveal the damage that occurs in the central nervous system of rat pups exposed to Aroclor 1254 in the prenatal period and to show the inhibiting effect of curcumin, which is a strong anti-oxidant and neuroprotective substance. Method: The study established 3 groups of adult female and male Wistar albino rats. The rats were mated within these groups and the offspring rats were evaluated within the group given Aroclor 1254 only (n=10) and the group was given both Aroclor 1254 and curcumin (n=10) and the control group (n=10). The groups were compared in respect of pathomorphological damage. The immunohistochemical evaluation was made of 8-hydroxdeoxyguanosine (8-OHdG), 4-hydroxynoneal (4HNE), myelin basic protein (MBP) expressions and TUNEL reaction. The biochemical evaluation was made of the changes in the TAS-TOS and Neuron Specific Enolase (NSE) levels. Damage was seen to have been reduced with curcumin in the 8OHdG and TUNEL reactions, especially in the forebrain and the midbrain, although the dosage applied did not significantly change TAS and TOS levels. Consequently, it was understood that Aroclor 1254 caused damage in the central nervous system of the pup in the prenatal period, and curcumin reduced these negative effects, particularly in the forebrain and the midbrain. Conclusion: It was concluded that curcumin could be a potential neuroprotective agent and would be more effective at higher doses.

Follicular Histomorphometry and Evaluation of Ovarian Apoptosis in Queens of Different Age Groups

Background: In humans and bitchs the age is another factor that may affect the size of ovarian structures, verifying alterations in the quality of the pool and size of follicular structures, which can compromise the use of these structures for in vitro maturation. There are no reports correlating the morphometric characteristics of the follicles and ovarian apoptosis at different ages in cats. The aim of this study was to evaluate the histomorphometric parameters of follicular growth and the relationship with the occurrence of apoptosis in ovarian tissue of young, adult and senile queens.Materials, Methods & Results: Eighteen domestic queens, multiparous, of different breeds and age groups were used in this study and divided into three groups according to their ages: five months to one year - young; (7.8 ± 1.0 months); one to six years - adults (2.8 ± 0.5 years); and more than six years - senile (8.0 ± 0.9 years). Vaginal cytology was performed in order to characterize the estrous phase associated with plasma concentrations of progesterone. The morphology and percentage of the vaginal epithelium cells were evaluated and queens were classified into estrous and non-estrus and plasma concentrations of progesterone were determined. Ovarian samples were collected after ovariohysterectomy to routine histological processin and all follicles were counted and categorized into two groups, non-atresic and atresic. The mean follicular and oocyte diameters were calculated between the measurement of the largest diameter and perpendicular diameter. The relationship between follicle and oocyte were determined using the measurements of diameter, area and perimeter. The apoptotic cells were detected and cells were considered positive when TUNEL reaction was detected. The morphometric index of 1039 follicles were evaluated. Primordial follicles in young animals showed larger diameter, follicular area and perimeter than the structures of adult queens, as well as the unilaminar primary follicles of the same group were larger compared with senile animals (P < 0.05). Comparing adult and younger queens, the first showed a significant decrease of oocyte diameter in primary and unilaminar primary follicles, as well as oocyte area and perimeter (P < 0.05). The values for follicular diameter, oocyte area and perimeter for multilaminar primary, secondary and pre-ovulatory structures did not present statistical differences between the groups (P > 0.05). For the pre-ovulatory follicles there was no positive correlation between the oocyte growths regarding the follicles (P > 0.05). Only in senile animals positive markers for apoptosis were identified in nuclei of primordial follicles. No significant differences concerning the number of follicles and Tunel positive cells were observed between groups (P > 0.05).Discussion: Considering the importance of this study for greater knowledge in the basic aspects for reproductive biotechnologies, we verified that secondary follicles showed the largest diameters and younger animals the largest values for diameter, area and perimeter, suggesting that this age group could be ideal for the use and manipulation of oocytes. The process of follicular atresia is characterized by the occurrence of apoptosis, or programmed cell death when the organism begins to efficiently eliminate dysfunctional cells. The study of follicular apoptosis in small animals, especially in cats, is very important for the development of reproduction biotechnologies. Phenomenon of apoptosis showed no relationship with age in queens, occurring in a physiological, continuous and proportionate manner considering the number of nondominant follicles involved in each estrous cycle.

170 IN VITRO EMBRYO PRODUCTION AND APOPTOSIS DETECTION USING BOVINE OOCYTES MATURED WITH IGF-I OR IGF-LongR3

The aim of this study was to compare the differential effects of the addition of IGF-LongR3 or insulin-like growth factor-1 (IGF-1), during in vitro maturation (IVM) of bovine oocytes on embryonic development, by analysing embryonic viability and apoptosis. Ovaries collected at a slaughterhouse were transported in saline solution (NaCl 0.9%) at 38°C. Follicles of 2–8 mm of diameter were aspirated. Only cumulus-oocyte complexes (COC) with homogeneous cytoplasm, surrounded by at least 3 layers of compact cumulus cells were selected in Dulbecco’ modified PBS 0.3% polyvinyl alcohol (PVA) and transferred to TCM HEPES at 38°C. Maturation medium was composed by TCM199, 0.2 mM pyruvate, 1 μg mL–1 FSH, 50 μg mL–1 LH, 100 μg mL–1 streptomycin sulfate, 100 UI mL–1 penicillin, and 85 μg mL–1 amikacin. Four experimental groups were determined using basic medium supplemented with fetal bovine serum (FBS; 10%), PVA (3 mg mL–1 Polyvinylpyrrolidone), IGF-1 (3.100 ng mL–1 insulin growth factor), LongR3-IGF (14.100 ng mL–1 long-chain human insulin growth factor 1-r3) respectively. The IVM was performed in Petri dishes (35 × 15 mm) with 90-μL droplets of the respective media, covered with sterile mineral oil, in 5% CO2 and 38.5°C temperature atmosphere for 22–24 h. The matured oocytes were fertilised with 2 × 106 spermatozoa mL–1 and incubated for 18 h. Embryos were denuded and cultivated in SOFaa medium (2.7 mM myoinositol, 0.2 mM pyruvate, 5 mg mL–1 BSA, 100 μg mL–1 streptomycin, 100 UI mL–1 penicillin, 85 μg mL–1 amikacin, and 25 μL mL–1 of FBS). Six repetitions were performed. After 7 days, blastocyst formation was analysed and all blastocysts were submitted to the TUNEL reaction. (In Situ Cell Death Detection Kit with Fluorescein, Roche®, Mannheim, BW, Germany), according to the technique adapted by Paula-Lopes and Hansen (2002).Green nuclei fluorescence (fluorescein isothiocyanate; FITC) was considered with fragmented DNA. Data were analysed by ANOVA), and least-squares means was used to verify differences of means using the PROC GLIMMIX model of SAS statistical software package (SAS Institute, Cary, NC, USA). Cleavage rate was similar for all groups. There was no statistical difference (P > 0.05) between groups regarding to embryo production. Most of the blastocysts obtained at Day 7 of culture had good quality (grade I). However, a difference (P = 0.03) on the number of expanded blastocyst was found between FBS (20.83 ± 3.22) and PVA (10.18 ± 3.22) groups. Furthermore, IGF-1 (12.03 ± 1.60) group showed a higher (P = 0.04, P = 0.02) apoptosis rates compared to groups FBS (7.96 ± 1.29) and PVA (6.97 ± 1.48). In the present study, it was observed that the addition of IGF-1 or LongR3-IGF during IVM provided a similar embryo production compared to FBS. On the other hand, embryos obtained from oocytes maturated on medium with the addition of IGF-1 had a high number of cells with fragmented DNA, indicating a more apoptosis.

243 THE SEX OF THE BOVINE EMBRYO AFFECTS APOPTOTIC RATES AT THE BLASTOCYST STAGE

Male and female pre-implantation bovine embryos may differ in several aspects such as kinetics of development, metabolism, or gene expression. These differences vary between culture conditions and may lead to shifts in sex ratio. In a previous study, we showed that female Day 7 blastocysts display a higher apoptotic rate than male ones when cultured in the presence of 5% FCS (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The difference was less important in the presence of BSA. This previous study was performed on in vitro-produced embryos produced with sexed semen and analysed using confocal microscopy. The objective of the present work is to confirm these differences using a) first, the unsexed semen of the same bull (bull 1) as the one used in the previous study in order to exclude any potential bias induced by the use of sexed semen and b) second, the unsexed semen of another bull (bull 2) in order to generalise our findings to the Bos taurus species. Levels of apoptosis were assessed in Day 7 blastocysts using immunohistochemical staining of cleaved caspase-3 and detection of fragmented DNA by TUNEL reaction on Day-7 blastocysts cultured with 5% FCS. Quantification of the number of stained cells was achieved with a fluorescence microscope. After cell counting, embryos were recovered and sexed by PCR. In both experiments, a higher proportion of cells showing caspase staining was observed in female (n = 145) than in male (n = 215) embryos (Bull 1: male: 11.8 ± 0.6%; female: 17.6 ± 1.1%, 2-way ANOVA, P ≤ 0.0001; bull 2: male: 9.5 ± 0.4%; female: 13.3 ± 0.6%, P ≤ 0.0001), whereas the proportion of TUNEL-stained cells was only significantly higher in female embryos produced with the semen of bull 2 (bull 1: male: 10.8 ± 0.4%; female: 11.4 ± 0.6%, P = 0.12; bull 2: male: 7.2 ± 0.3%; female: 9.1 ± 0.4%, P = 0.0009). A significant difference in cell number was observed between male and female blastocysts produced with the semen of bull 2 (male: 172 ± 5; female: 154 ± 5, P = 0.02) and the same tendency was observed for embryos generated with the semen of bull 1 (male: 143 ± 4; female: 132 ± 4, P = 0.07). In conclusion, our study demonstrated that the use of sexed semen does not interfere with the pattern of caspase and TUNEL staining previously observed. Moreover, a similar pattern was observed with 2 different bulls. We can thus conclude that the level of apoptosis of bovine Day 7 blastocysts produced in the presence of FCS is higher in female than male embryos. This could be related to the tendency towards a lower cell number in female blastocysts and to the shift in sex ratio in favour of male embryos often observed in the presence of serum.

Correlation between expressions of hypoxia -inducible factor (HIF-1α), blood vessels density, cell proliferation, and apoptosis intensity in canine fibromas and fibrosarcomas

The study aimed to demonstrate the expression of hypoxia-inducible factor (HIF-1α) in soft tissue mesenchymal tumours (fibroma and fibrosarcoma) in dogs. An attempt was made to correlate the obtained results with density of blood vessels (expression of von Willebrand Factor, vWF), expression of Ki-67 proliferation antigen, and with intensity of apoptosis in studied tumours. The study was performed on paraffin sections of 15 fibromas and 40 fibrosarcomas sampled from 55 female dogs aged 6 to 16 years. Immunohistochemical staining against HIF-1α, vWF, and Ki-67 was performed. Apoptosis was detected with the use of TUNEL reaction. A significantly higher HIF-1α expression was noted in fibrosarcomas in comparison to fibromas (P < 0.0001). HIF-1α expression in fibromas manifested strong positive correlation with tumour vascularity (r = 0.67, P = 0.007). Moreover, HIF-1α expression in fibrosarcomas manifested a moderate positive correlation with tumour malignancy grade (r = 0.44, P = 0.004), tumour vascularity (r = 0.52, P < 0.001), Ki-67 antigen expression (r = 0.42; P = 0.007), and TUNELpositive cells (r = 0.37, P = 0.017). Expression of HIF-1α was detected in 86.7% of fibroma type tumours and in 100% of fibrosarcomas. In all studied tumours expression of HIF-1α manifested positive correlation with the density of blood vessels, and in fibrosarcomas it correlated also with malignancy grade, intensity of Ki-67 expression, and with intensity of apoptosis in tumour cells.

161 USE OF FORSKOLIN TO DELAY MEIOSIS AND PRODUCE IN VITRO BOVINE EMBRYOS

The maintenance of oocytes in germinal vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology. This study aimed to show if the use of forskolin is able to inhibit and reverse the maturation in bovine oocytes, producing a higher rate of in vitro embryos without apoptosis rates. Eight replicates in total were performed. Nellore oocytes were matured in TCM-199 and to delay meiosis, the oocytes (n = 584) were maintained for 6 h in medium in presence of 0.025, 0.05, or 0.1 mM Forskolin. Then, the oocytes were cultured for 18 h in agent-free medium to resume meiosis. After resumption of meiosis, the oocytes (n = 336) were stained with Hoechst 33342 to evaluate the state of the nucleus: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), or degenerated or unidentified (D/U). Then (Day 0) oocytes were fertilized in human tubal fluid (Irvine, New Zealand) and the presumed zygotes were culture in SOFaa + 0.6% BSA + 2.5% FCS until Day 7, when the blastocyst (n = 177) rate was evaluated. Apoptosis in blastocysts was assessed by terminal deoxynucleotidyl transferase uracil nick-end labeling (TUNEL) reaction. Data were analysed by ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. There were no statistical differences in state of the nucleus, only in MI (Control = GV: 0.0, GVBD: 0.8, MI: 8.3a, MII: 67.7, D/U: 7.3; F 0.025 mM = GV: 2.8, GVBD: 0.7, MI: 20.8ab, MII: 67.7, D/U: 8.9; F 0.05 mM = GV: 0.0, GVBD: 4.4, MI: 15.8ab, MII: 65.9, D/U: 13.7; and F 0.1 mM = GV: 0.0, GVBD: 1.0, MI: 34.1b, MII: 50.2, D/U: 14.6; P < 0.05). There were no statistical differences in blastocyst rate (Control: 36.7, F 0.025 mM: 32.6, F 0.05 mM: 29.2 and F 0.1 mM: 25.1 – P > 0.05). But when we analysed the apoptosis rate, differences were found among groups: (Control: 12.1a, F 0.025 mM: 12.9a, F 0.05 mM: 13.5a and F 0.1 mM: 30b; P < 0.05). Although Forskolin was able to inhibit meiosis and produce embryos at the same rates as controls, the higher dosage of this drug damaged the embryos. The authors acknowledge FAPESP 10/50410-2 for support.