Postnatal Rat Liver Research Articles

Immunohistochemical examination of p97/VCP expression in developing mouse pancreas and liver

Abstract
 Aim: The 97-kDa Valosin-containing protein has important functions in proteolysis. Although the expression of p97 / VCP has been studied in many types of cells and tissues, the localization of p97 / VCP at cellular level in developing mouse pancreas and liver has not been determined. Therefore, the aim of our study was investigate the immuolocalization of p97/VCP in the rat fetal and postnatal liver and pancreas. 
 Material Methods: The liver and pancreas from fetal, postnatal (1, 5, 15, 35 days old ) and adult (50 days old) mice were examined by using immunohistochemistry in order to determine the expression of p97/VCP. Furthermore the development of mouse pancreas and liver were histomorphologically analyzed under light microscope. 
 Results: The histological structures of the mouse pancreas and liver were maintained in postnatal period. The histological differences between endocrine and exocrine pancreas were significantly observed from the postnatal 5th day. The expression of p97/VCP in Langerhans islets was determined from day 5. p97/VCP expression was also seen in the exocrine pancreas in all postnatal days. p97/VCP was expressed in developing pancreatic and liver tissues and its expression was increased with the development.
 Conclusion: This study is the first to examine the distribution and the localization of p97/VCP in the fetal and postnatal rat liver and pancreas. This study forms the basis for clinical studies in order to determine the function of p97 / VCP in liver and pancreatic cells.

Effect of Administration of Acrylamide and Possible Protective Role of Vitamin E on Postnatal Rat Liver Structure

Effect of Administration of Acrylamide and Possible Protective Role of Vitamin E on Postnatal Rat Liver Structure

Effects of parental folate deficiency on the folate content, global DNA methylation, and expressions of FRα, IGF-2 and IGF-1R in the postnatal rat liver

We examined the effect of parental folate deficiency on the folate content, global DNA methylation, folate receptor-alpha (FRα), insulin-like-growth factor-2 (IGF-2) and -1 receptor (IGF-1R) in the liver and plasma homocysteine in the postnatal rat. Male and female rats were randomly fed a folic acid-deficient (paternal folate-deficient, PD and maternal folate-deficient, MD), or folic acid-supplemented diet (paternal folate-supplemented, PS and maternal-folate-supplemented, MS) for four weeks. They were mated and grouped accordingly: PSxMS, PSxMD, PDxMS, and PDxMD. Pups were killed on day 21 of lactation. The hepatic folate content was markedly reduced in the PDxMD and PSxMD and PDxMS as compared with the PSxMS group. The hepatic global DNA methylation was decreased in the PDxMS and PSxMD groups as much as in the PDxMD group, and all the three groups were significantly lower as compared to the PSxMS group. There were no significant differences in the hepatic FRα, IGF-2 and IGF-1R expressions among the groups. Positive correlations were found between the hepatic folate content and global DNA methylation and protein expressions of FRα, IGF-2 and IGF-1R, whereas an inverse correlation was found between hepatic folate content and plasma homocysteine level in the 3-week-old rat pup. The results of this study show that both paternal and maternal folate deficiency at mating can influence the folate content and global DNA methylation in the postnatal rat liver.

Efficacy of melatonin on offspring liver maturation in pinealectomized pregnant rats subjected to experimental epilepsy

In clinical practice, maternal epilepsy is a disabling disease for newborn infants, but current data concerning the effect of epileptic phenomena in pregnant mothers on newborns are still limited. This study was undertaken to investigate the effects of pinealectomy (Px) and melatonin treatments on the morphological changes in the liver tissue of newborn rats following experimental epilepsy during pregnancy. Female Swiss Albino rats were divided into five groups: intact control group; saline control group; epilepsy group; epilepsy plus Px group; and melatonin-treated epilepsy plus Px group. At one month after Px, an acute grand mal epileptic seizure was induced by penicillin-G during their pregnancy in all animals except the control groups. On the neonatal first day, newborn rats were perfused with intracardiac fixative solution, and then livers were removed and processed for toluidine blue, periodic acid-Schiff (PAS) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reactivity. Normal migration and hepatic maturation were determined in the postnatal rat liver in the control groups, while the morphological structure of the liver in the epilepsy and epilepsy plus Px groups corresponded to the early embryonic period. In the melatonin-treated epilepsy plus Px group, the number of TUNEL positive cells decreased significantly compared to both epilepsy and epilepsy plus Px groups; however, there was no statistically significant difference from the control groups as a result of melatonin activity. Some histological findings consistent with chronic fetal distress in newborns of mother rats with epilepsy and Px were observed. Melatonin could be a candidate protective drug for the development of liver tissue in pregnant patients with epilepsy.

Developmental Changes of Aldehyde Oxidase in Postnatal Rat Liver

In this study, the developmental changes and variability of aldehyde oxidase in postnatal rat liver were examined. Postnatal day 1, 7 and 14 rats showed little or no liver aldehyde oxidase activity, as evaluated in terms of the activities for oxidation of benzaldehyde to benzoic acid, N-1-methylnicotinamide (NMN) to N-1-methyl-2-pyridone-5-carboxamide (2-PY) and N-1-methyl-4-pyridone-3-carboxamide (4-PY), and methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX). However, these oxidase activities were markedly increased in liver cytosol from the rats after postnatal day 14. The activity was then maintained up to 6 weeks. The amounts of 2-PY and 4-PY formed from NMN were almost the same. The development of aldehyde oxidase activity toward benzaldehyde was closely correlated with that of oxidase activity toward NMN and MTX. The expression of aldehyde oxidase at postnatal day 14 was confirmed by Western blotting analysis. The density of bands of aldehyde oxidase was closely correlated with the oxidase activity toward benzaldehyde. The developmental changes of aldehyde oxidase activities during postnatal reflected the changes in the amount of the oxidase protein. Thus, aldehyde oxidase activity in rats rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated by expression of the protein.

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X6 receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X4 and P2X6 receptor subunits and dendritic cells co-expressed P2X4 and P2X7 receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X6 receptor on Kupffer cells, suggesting that the P2X6 receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.

Two Isoforms of Trimming Glucosidase II Exist in Mammalian Tissues and Cell Lines but Not in Yeast and Insect Cells

We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

Hepatic jagged1 expression studies.

Mutations in Jagged1, a Notch ligand, have been shown to result in Alagille syndrome (AGS), however, the causal link between haploinsufficiency of Jagged1 and intrahepatic ductal paucity is unknown. This survey was performed to determine the expression pattern of Jagged1 in the fetal and postnatal liver. Reverse transcription polymerase chain reaction (RT-PCR) showed Jagged1 expression in all samples studied including rat liver embryonic days 16 to 21, 1-day-old, 1-week-old, and 2-month-old adult rats. RT-PCR detected Jagged1 in total liver RNA extracted from cadaver organ donor samples from reduced human grafts and explanted native livers from a variety of pediatric disorders including AGS, biliary atresia, congenital hepatic fibrosis, sclerosing cholangitis, cystic fibrosis, fulminant hepatic failure, tyrosinemia, and chronic rejection. Immunohistochemistry showed Jagged1 expression in human fetal samples localized to the ductal plate from 14-week gestation onward. Expression in the postnatal liver was seen in biliary epithelium and zone 3 hepatocytes. In conclusion, these studies show that Jagged1 is expressed in the fetal and postnatal liver in health and disease. We show localization of expression by immunohistochemistry to ductal plate epithelium in human fetal samples and to the biliary epithelium and zone 3 hepatocytes in human postnatal samples. Our results show the localization of Jagged1 in fetal liver and demonstration of Jagged1 expression in postnatal rat and human liver specimens. Further studies of Jagged1 and the Notch signaling pathway are expected to elucidate mechanisms of the regulation of biliary epithelial growth and development.

Immunohistochemical Detection of Glutathione S-transferase P in Postnatal Rat Liver after Administration of Dexamethasone.

The localization of glutathione S-transferase P (GST-P) in postnatal rat liver was examined by an immunohistochemical technique. Dexamethasone (DEX) was injected intraperitoneally into postnatal rats at a dose of 2 μg/g body weight daily for 4 days. GST-P was detected only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 8-day-old rats that did not receive DEX-administration. In contrast, dispersed-positive hepatocytes were observed in the liver lobules of rats that were administered DEX from 4 days after birth. In these rats, GST-P was found in the cytoplasm of those hepatocytes and epithelial cells of the interlobular bile ducts. GST-P was observed only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 13-day-old rats that did not receive DEX-administration and those that began DEX-administration at 9 days. The immunoreactivity was stronger compared to the 8-day-old rats. These results indicate that DEX promotes the appearance of hepatocytes that contain the GST-P protein in early postnatal rat liver.

Measurement of GLUT mRNA in Liver of Fetal and Neonatal Rats Using a Novel Method of Quantitative Polymerase Chain Reaction

Transfer of glucose into the hepatocyte is mediated by glucose transporters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.

Developmental Expression of the Neurotensin Gene in the Rat Liver

This study determined whether the neurotensin gene is expressed during early development of the liver. Neurotensin (NT), a gut tridecapeptide localized mainly to the distal small bowel and brain of adults, is an important hormone regulating gut motility, secretion and mucosal growth. Expression of NT peptide and the gene is found in fibrolamellar hepatocarcinomas, a variant of hepatocellular carcinoma, but not in the normal adult liver. Northern and in situ hybridization techniques were used to determine expression of the neurotensin gene (NT/N) in the normal developing liver. NT/N is expressed in the fetal and early postnatal rat liver, but expression is repressed in the liver of the adult. In situ hybridization confirms the authors' Northern data and demonstrates a random distribution of NT/N expression in the fetal and 3-day postnatal liver. The authors conclude from this study that NT/N is expressed during early development of the rat liver with subsequent repression in the adult. NT/N may be reexpressed with malignant transformation of the liver.

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

Abstract To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5–21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.

Transcription of the insulin-like growth factor-binding protein-2 gene is increased in neonatal and fasted adult rat liver.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that specifically bind IGF-I and IGF-II, determine their bioavailability to tissues, and modulate their actions in target tissues. Levels of IGFBPs in plasma and IGFBP mRNAs in liver are highly regulated with developmental age and metabolic status. We now demonstrate that the increase in IGFBP-2 mRNA in fasted adult rat liver and in the liver of normal neonatal rats reflects an increased rate of transcription. When adult rats were fasted for 2-3 days, IGFBP-2 mRNA was increased in liver, but not in brain or kidney. The increase in hepatic IGFBP-2 mRNA was observed after only 1 day of fasting. Levels decreased by half after 6 h of refeeding and returned to their low starting values after 2 days of refeeding. Transcription-elongation experiments indicated that transcription of the IGFBP-2 gene was increased in fasted liver. The rate of transcription increased 9.2- +/- 3.5-fold for transcripts labeled in exon 1 and 6.6- +/- 2.4-fold for transcripts labeled in exons 2, 3, and 4, suggesting that fasting causes a uniform increase in the number of RNA polymerase II molecules along the length of the IGFBP-2 gene. We infer from these results that the regulation of IGFBP-2 gene transcription in fasting occurs at the level of initiation rather than elongation. IGFBP-2 gene transcription also was increased 3.8- +/- 1.2-fold (exon 1) and 2.9- +/- 0.9-fold (exons 2, 3, and 4) in nuclei from 2-day postnatal rat liver compared with adult rat liver, consistent with the greater abundance of IGFBP-2 mRNA in neonatal rat liver.

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.

1,2-Diacylglycerol and ceramide levels in rat liver and skeletal muscle in vivo

1,2-Diacylglycerol and ceramide levels were measured in liver and skeletal muscle of rats under the following four experimental conditions: 1) during rapid fetal growth in the second half of gestation and during postnatal aging, 2) during tissue anoxia lasting up to 10 min, 3) during fasting for up to 6 days, and 4) during stress induced by injection of endotoxin (lipopolysaccharide from Salmonella enteritidis). 1,2-Diacylglycerol and ceramide levels in fetal liver were 77 and 58% lower, respectively, than those of young rats after weaning. 1,2-Diacylglycerol and ceramide concentrations in liver of postnatal rats were not influenced by aging. Anoxia produced a 41-64% increase in liver 1,2-diacylglycerol levels and a 9-21% increase in liver ceramides. Fasting of adult rats for up to 6 days had no effect on 1,2-diacylglycerol and ceramide levels in liver. However, fasting increased skeletal muscle concentrations of 1,2-diacylglycerol and ceramides 88 and 44%, respectively. Injection of endotoxin had a biphasic effect on liver 1,2-diacylglycerol levels, causing a transient 52% increase at 1 h, followed by a 24-38% decrease below the control level at 17 h after endotoxin administration. Liver ceramide levels were increased 66, 52, and 27% at 1, 3, and 5 h after endotoxin, respectively, but did not differ from control at 17 h after injection. Endotoxin had no effect on muscle 1,2-diacylglycerol and ceramide concentrations at any interval.

Morphological and immunohistochemical assessment of intrahepatic bile duct development in the rat

The hypotheses that intrahepatic bile ducts are derived either by a transformation of periportal liver cells or by dichotomous branching of the extrahepatic bile ducts were investigated in fetal and postnatal rat livers by histological and immunohistochemical methods using an antiserum to prekeratin which, in the liver, binds to biliary epithelial cells (BEC). In conventionally stained sections, bile duct development was observed to begin in the 19 day fetus around the larger branches of the portal vein, with the formation of lumina surrounded by cuboidal or elongated hepatoblast-like cells on the portal aspect and readily distinguished hepatoblasts on the lobular aspect. At 21 days, these structures had developed into canals of Hering lined jointly by recognizable liver cells and BEC. The number of canals of Hering per portal tract peaked at 22 days' gestation and diminished in number at birth and over the ensuing 56 h, with a concomitant increase in fully formed ducts. Bile ducts lined completely by BEC were first found at 20 days. Immunohistochemically, prekeratin antigens were first detected at 20 days in duct-like structures not only in phenotypic BEC but also in adjacent cells with an hepatoblast phenotype. Such intermediate cells were present until birth. These findings support the view that intrahepatic bile ducts develop by a reorganization and modulation of the periportal hepatoblasts to BEC.

Topogenesis of glycogen distribution pattern in post-natal rat liver in reference to the activity-rest cycle

The present study, on immature rats, revealed that in the liver with exclusively diploid cell population (MD), the glycogen content was half of the adult level and the 24 h distribution pattern was reverse, i.e. highest in the evening and lowest in the morning. In these animals, in general, the protein content was high and did not show any circadian rhythm but incidentally showed a time dependent zonal distribution pattern. The low glucose-6-phosphatase activity reciprocates with low glycogen content. The relative number of cells per unit area showed a time dependent distribution pattern. The liver with equal distribution of diploid and tetraploid classes (MD:MT), attained the classical circadian rhythm of glycogen with high morning and low evening oscillation pattern. The TRIDENT measurements manifest the zonal distribution pattern with high values at the perilobular region (PL) and low at the centrolobular region (CL). The relative cell number study along the acinus demonstrated more number of cells around the PL region than that at CL region, indicating a variation in the cell size. Eventually, the protein content showed a circadian rhythm in this group, with high amount in the morning. The zonal distribution pattern always revealed the high content near the perilobular region. This could be due to more number of cells per unit area in this region. The glucose-6-phosphatase showed a circadian rhythm. The typical high glucose-6-phosphatase in the perilobular region could be further subzonated into small groups of high activity surrounded by lighter zones, thus establishing the heterogeneous function of liver parenchymal cells.

Differential changes of nuclear-envelope-associated enzyme activities involved in nucleocytoplasmic mRNA transport in the developing rat brain and liver

Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3′poly(A) tail of poly(A) + mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A) − mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A) − mRNA unique in the brain.

Growth and polyploidization of the liver of early postnatal rats treated with blfomycin

Bleomycin was administered to 6- and 10-day-old rats and its effects were examined after a two-week survival period. Decreases in body weight and some phenotype abnormalities occurred in both age groups. Decreased relative liver weight (g/g b.w.) was observed only in animals injected on PD 10. DNA microdensitometry and microfluorometry (Feulgen reaction and Hoechst 33342 staining) revealed an increase in the frequency of intermediate DNA values in animals injected on PD6 and killed on PD20. In older animals, i.e. in rats injected on PD10 and killed on PD25, increases in 4c mononucleate and 8c binucleate cells and also a reduction of the relative number of binucleate cells appeared, as compared with age-matched controls. In both age groups, karyological abnormalities, including micronucleate cells and imbalanced DNA content in binucleate cells, were observed.