The morphofunctional state of the fetal thyroid gland in maternal-fetal infections caused by Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae

Aim – experimental modelling and determination of the morphofunctional state of the thyroid gland of a rat fetus in maternal-fetal infections caused by E. coli, S. aureus and K. pneumoniae. Material and methods. We conducted a controlled experimental study with block randomization on 85 female Wistar Albino Glaxo rats, which, depending on the group assigned randomly, were infected or not with E. coli, S. aureus and K. pneumoniae before pregnancy with subsequent pathomorphological examination of 37 thyroid glands obtained from rats’ fetuses. The set of methods was applied: histological – HE and Mallory staining; indirect immunofluorescence: moAb to T4, types I and III collagen, IL-6, and TNF; histomorphometry using ImageJ software. Statistical analysis was performed with the R environment and packages “ggplot”, “dplyr”, “pastecs”, “graphics” for Shapiro–Wilk test, Bartlett’s test, Box-Cox method, ANOVA, and plotting. The null hypothesis was rejected in cases when an error probability (P) did not exceed the type I error set at 0.001 (P < 0.001). Results. In the thyroid glands of fetuses obtained from mothers infected with E. coli, S. aureus and K. pneumoniae, in comparison with the control group, a statistically significant increase in the following morphofunctional parameters was determined: the diameter and area of the follicle, the area of the colloid, the height and area of the thyrocyte, the intensity and area of the fluorescence of the follicular thyrocytes and colloid, in samples labeled MoAb to T4, the intensity of the fluorescence of collagen types I and III; a decrease in the area of the nucleus in relation to the area of the cytoplasm, which was reflected in a decrease in the NCR index. The most pronounced differences in morphological and functional parameters from the norm were found in the thyroid gland of fetuses from mothers infected with S. aureus. Conclusions. The revealed changes in the morphofunctional parameters of the thyroid gland of a rat fetus, which experienced the influence of maternal-fetal infections caused by E. coli, S. aureus and K. pneumoniae, are unidirectional and statistically significantly different from those recorded during physiological organogenesis. They correspond to an increase in the gland’s secretory activity and indicate the acceleration of organ’s maturation.

ALPHA-PINENE ATTENUATES MICROGLIAL NF-ΚB ACTIVATION AND INOS EXPRESSION IN GP120-INDUCED NEUROINFLAMMATION

Background: Neuro-inflammation plays a role in the pathogenesis of HIV-associated dementia (HIV). Activation of microglia is essential for triggering inflammatory-mediated neurotoxicity. HIV-1 120 kDa envelope glycoprotein (gp120) induces microglial NF-κB signaling which in turn induce pro-inflammatory and iNOS gene transcription. Continuous or excessive activation of NF-κB signaling lead to persistent production of TNF-α and nitric oxide by microglia and induce neuronal apoptosis. Alpha-pinene is a natural substance found in pine tree and has efficacy on inhibiting NF-κB signaling. Objective: This study was designed as a true experimental study and aimed to investigate the effect of alpha-pinene administration toward inflammatory response represented by the percentage of microglia containing activated NF-κB and iNOS expression. Methods: Neuron-glia primary culture from brain tissue of rat fetus was divided into 5 groups as follows: negative control; positive control (gp120 1nM); treatment I, II, and III (gp120 1 nM + alpha-pinene 0.4 µg/mL, 2 µg/mL, and 10 µg/mL, respectively). Microglial NF-κB and iNOS expression were analyzed using immunohistochemistry method. Neuronal apoptosis was measured by TUNNEL method. Results: Result showed that alpha-pinene administration on gp120-treated neuron-glia at all dosages decrease NF-kB activation, iNOS expression, and apoptotic neuron significantly as compared to the gp120-only treated group (p<0.05). Furthermore, alpha-pinene did not affect NF-kB activation and neuronal apoptosis (p>0.05), but significantly elevate iNOS expression (p<0.05) mainly in dosage I and II. Conclusion: We concluded that alpha-pinene has neuroprotective effect on gp120-treated neuron-glia cells through modulation of NF-kB and iNOS expression thus inhibit neuronal apoptosis. Keywords: apoptosis, gp120, iNOS, neuroinflammation, NF-kB

Alpha-lipoic acid ameliorates cytarabine-induced developmental anomalies in rat fetus

Cytarabine (Ara-C) is a nucleoside analogue used in the treatment of cancers and viral infections. It has teratogenic potential and causes a variety of birth defects in fetuses. Alpha-lipoic acid (ALA) is a natural antioxidant offers protection against the developmental toxicity induced by drug- or toxicant-exposure or pathological conditions. This study was aimed at evaluating the protective effect of ALA against Ara-C induced developmental toxicity in rat fetus. Pregnant rats divided into five groups and received normal saline, ALA200 mg/kg, Ara-C12.5 mg/kg, Ara-C25 mg/kg and, Ara-C25 mg/kg plus ALA200 mg/kg respectively from gestational day (GD) 8 to GD14 and sacrificed on GD21. Ara-C treatment led to a significant and dose-dependent decrease in food intake, weight gain, placental weight, and an increase in oxidative stress in pregnant rats. Further, the in-utero exposure to Ara-C led to an increase in fetal mortality, resorptions, oxidative stress, external morphological anomalies and limb abnormalities, and impaired ossification. Co-administration of ALA resulted in amelioration of the footprints of Ara-C induced toxicity in pregnant rats as well as the fetus. These findings indicate that the ALA supplementation offers protection against developmental toxicity caused by Ara-C prenatal exposure in rats.

WHAT IS THE EFFECTS OF PARACETAMOL APPLIED AT DIFFERENT DOSES TO HEAVY METAL LEVELS IN RAT FETUS?

Paracetamol is the first preferred pharmacological agent as a pain reliever and antipyretic in all periods of pregnancy. In this study, we aimed to analyze trace element and heavy metal levels in the placenta, intestinal and kidney tissues of rats in the early development period of paracetamol. Sixteen pregnant rats were randomly divided into four groups; the control group, the 50 mg/kg paracetamol group, the 250 mg/kg paracetamol group, the 500 mg/kg paracetamol group. There was a statistically significant decrease in the placental weight of the experimental groups compared to the control group. However, a statistically significant difference was found in terms of cobalt (Co) and lead (Pb) levels compared to the control group and the groups that received various doses of paracetamol. There were also statistically significant differences in intestinal chromium (Cr), selenium (Se) and cadmium (Cd) levels in the studied groups. In addition, significant differences were detected in all trace elements and heavy metal levels except Cd in the groups studied in kidney tissue (p <0.01 for all). As a result, it was determined that the use of paracetamol during pregnancy disrupted the current balance due to the increase in dose. In addition, it was observed that the weight of the placenta decreased due to the paracetamol dose, and the placenta Pb and Co levels increased. In other tissues, there was no toxic concentration at heavy metal and trace element levels, but the highest levels were determined in the control group.

In utero xenotransplantation of mice bone marrow-derived stromal/stem cells into fetal rat liver: A preliminary study

Background: Animals can play an important role in preparing tissues for human through the development of xenotransplantation protocols. The most common problem with liver transplantation like any other organ transplantation is organ supply shortage. Objective: To evaluate the in utero xenotransplantation of mouse bone marrow-derived stromal/stem cells (BMSCs) to the liver of rat fetus to produce mouse liver tissue. Materials and Methods: BMSCs were isolated and confirmed from enhanced green fluorescent protein (eGFP)-genetic labeled mice. Using a microinjection protocol, mice BMSCs were injected into the liver of rat fetuses in utero on day 14 of pregnancy. After birth, livers were collected and the presence of mice eGFP-positive cells in rat livers was evaluated through polymerase chain reaction. Results: The eGFP mRNA was detected in the liver of injected infant rats. BMSCs of adult mice were capable to remain functional probably as hepatocyte-like cells in liver of infant rats after in utero xenotransplantation. Conclusion: BMSCs have the potential for intrauterine xenotransplantation for the treatment of liver dysfunction before birth. This method can also be used for xenoproduction of liver tissue for transplantation. Key words: Xenotransplantation, Liver, Bone marrow, Stromal/stem cell, Murine.

THE EFFECT OF LIMAN LEAVES EXTRACT ON FETAL RAT DEVELOPMENT

Objective: The objective of the study was to observe the effect of liman leaves extract on fetal rat development and provide safety information for its use during pregnancy. Methods: Estrus cycles of female Wistar rats were observed and mated to male rats, 0 day of pregnancy determined after finding a vaginal plug or in a vaginal smear, there was sperm. Sample was administered orally using sonde with a single administration on the 11th day of pregnancy, at doses of 3750 mg/kg bw, 1185 mg/kg bw, and 375 mg/kg bw. On the 19th day of pregnancy, the rats were sacrificed and then observed the number of implantation, corpus luteum, intrauterine death, and fetal abnormalities. Results: The dose of 375 mg/kg bw exposed the highest average implantation rate (13.20%) and the largest number of corpus luteum (13.90%). The highest total intrauterine death was presented by dose of 3750 mg/kg bw and significantly different (p<0.05) compared to the control group. The dose of 375 mg/kg bw expressed the highest percentage of embryos with resorption (19.03%), while the lowest average fetal body was shown by 3750 mg/kg bw compared to the other groups. The highest percentage of external abnormalities was given by the dose of 375 mg/kg bw (12.91%), which abnormalities found were dwarf, cleft palate, hydrocephalus, short sleeve, and hematoma. Conclusion: Liman leaves extract was mild teratogenic on Wistar rat fetus.

Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations

Background Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes. Purpose This study was aimed to investigate the optimal reference genes for PCR in ARM rat models. Methods We selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper). Results The abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs. Conclusions The most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.