Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1ß, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Appearance of Heterogeneous Macrophages During Development of Isoproterenol-Induced Rat Myocardial Fibrosis.

Macrophages appearing in lesions are polarized toward M1 (for inflammation) and M2 (for anti-inflammation/fibrosis) types. We analyzed immunophenotypes of macrophages appearing in myocardial lesion in rats injected once with isoproterenol (10 mg/kg body weight). Inflammation following myocardial necrosis on day 1 was seen with a peak on days 3 and 5, and thereafter, reparative fibrosis developed on days 7 to 28. CD68+ M1 macrophages were seen in the early stages of injury and inflammatory on days 1 to 7, and thereafter, CD163+ M2 macrophages increased in the late stages of fibrosis on days 7 to 28. There was the polarization of M1 and M2 macrophages. The kinetics of macrophages reacting to Iba-1 and Galectin-3 was similar to that of M1 macrophages, indicating that Iba1- and Gal-3-positive macrophages might have functions of M1 type. Double immunofluorescence revealed that CD204- and MHC class II-positive macrophages are polarized toward M1 and M2 types, respectively. CCR2 messenger RNA expression is transiently elevated on day 1. Since CCR2 is a marker of blood monocytes, M1 macrophages might be recruited from blood monocytes. Collectively, macrophages expressing heterogeneous immunophenotypes participate in myocardial fibrosis. These findings would be useful for understanding the pathogenesis of myocardial fibrosis and analyzing myocardial toxicity.

Characterization of Macrophages and Myofibroblasts Appearing in Dibutyltin Dichloride-Induced Rat Pancreatic Fibrosis.

Macrophages and myofibroblasts are important in fibrogenesis. The cellular characteristics in pancreatic fibrosis remain to be investigated. Pancreatic fibrosis was induced in F344 rats by a single intravenous injection of dibutyltin dichloride. Histopathologically, the induced pancreatic fibrosis was divided into 3 grades (1+, 2+, and 3+), based on collagen deposition. Immunohistochemically, CD68-expressing M1 macrophages increased with grade and CD163-expressing M2 macrophages also increased later than M1 macrophage appearance. Double immunofluorescence showed that there were macrophages coexpressing CD68 and CD163, suggesting a possible shift from M1 to M2 types; similarly, increased major histocompatibility complex class II- and CD204-expressing macrophages were polarized toward M1 and M2 types, respectively. These findings indicated the participation of M1- and M2-polarized macrophages. Mesenchymal cells staining positive for vimentin, desmin, and α-smooth muscle actin (α-SMA) increased with grade. There were mesenchymal cells coexpressing vimentin/α-SMA, desmin/α-SMA, and glial fibrillary acidic protein (GFAP)/α-SMA; Thy-1-expressing immature mesenchymal cells also increased in fibrotic lesions. Because α-SMA expression is a reliable marker for myofibroblasts, α-SMA-expressing pancreatic myofibroblasts might be originated from GFAP-expressing pancreatic stellate cells or Thy-1-expressing immature mesenchymal cells; the myofibroblasts could simultaneously express cytoskeletal proteins such as vimentin and desmin. The present findings would provide useful information for analyses based on features of macrophages and myofibroblasts in chemically induced pancreatic fibrosis.

Macrophage Populations and Expression of Regulatory Inflammatory Factors in Hepatic Macrophage-depleted Rat Livers under Lipopolysaccharide (LPS) Treatment.

To investigate the significance of the appearance of hepatic macrophages and expression of inflammatory factors in normal and macrophage-depleted livers, hepatic macrophages were depleted with liposome (Lipo)-encapsulated clodronate (CLD; 50 mg/kg, i.v.) followed by lipopolysaccharide (LPS) administration (0.1 mg/kg, i.p.) in F344 rats (CLD + LPS). Vehicle control rats (Lipo + LPS) received empty-Lipo before LPS. The low dose of LPS did not result in microscopic changes in the liver in either treatment group but did modulate M1 and M2 macrophage activity in Lipo + LPS rats without altering repopulating hepatic macrophages in CLD + LPS rats. LPS treatment in Lipo + LPS rats dramatically increased the M1 (IL-1ß, IL-6, TNF-α, and MCP-1) but not M2 macrophage-related factors (IL-4 and CSF-1) compared to CLD + LPS rats. In the CLD + LPS rats, the M2 macrophage-related factors IL-4 and CSF-1 were elevated. In conclusion, low-dose LPS activated hepatic macrophages in rat livers without causing liver injury or stimulating repopulating hepatic macrophages. These data suggest that LPS may alter the liver microenvironment by modulating M1 or M2 macrophage-related inflammatory mediators and macrophage-based hepatotoxicity.

Expression of ß-catenin in regenerating renal tubules of cisplatin-induced kidney failure in rats.

BACKGROUND: ß-Catenin is a multi-functional protein involved in nephrogenesis and also plays important roles in renal injury. Here, the expression of ß-catenin was investigated in the proximal renal tubular epithelial cells in cisplatin (CDDP)-induced acute kidney injury (AKI) and chronic kidney injury (CKI), because CDDP-induced renal lesions were characterized by proximal renal tubular epithelial degeneration/regeneration and subsequent interstitial fibrosis. METHODS: F344 rats were treated with CDDP. The expression of ß-catenin and proliferative (Ki67) or fibrogenic [vimentin, α-smooth action (α-SMA)] markers was analyzed by immunolabeling. RESULTS: ß-Catenin, vimentin and Ki67 were not seen in the proximal renal tubules of control rats. Interestingly, in CDDP-induced AKI, the regenerating proximal renal tubular epithelial cells reacting strongly with Ki67 expressed membranous or cytoplasmic ß-catenin and also showed a positive reaction to vimentin but not to α-SMA. In CDDP-induced CKI, the epithelial cells of abnormally dilated or atrophied renal tubules did not react to ß-catenin or Ki67, but showed positive reactions to vimentin and α-SMA. ß-Catenin mRNAs were significantly increased in AKI and significantly decreased in CKI. CONCLUSION: Newly expressed ß-catenin in the proximal renal tubules after AKI may participate in functional regeneration. In CKI, epithelial cells of abnormal renal tubules did not express ß-catenin but reacted to vimentin, and α-SMA might indicate the epithelial-mesenchymal transition (EMT) formation, because α-SMA is usually expressed in myofibroblasts forming via EMT. The presence or absence of ß-catenin expression would become a marker for the EMT phenomenon in progressive renal fibrosis.

Immunohistochemical analyses of the kinetics and distribution of macrophages in the developing rat kidney.

Macrophages are required during kidney development and appear in the initiation and propagation of renal injury. To establish baseline data, we analyzed the kinetics of the macrophage with different immunophenotypes in the developing rat kidney (fetus at 18 and 20 days, neonate at 1-21 days, and adult at 7-weeks old). Macrophages reacting to CD68, CD163, and MHC class II were identified in the cortex and medulla of the developing rat kidney. CD68+ macrophages appeared in the fetal kidney as early as fetal day 18, and the number increased gradually in the neonatal kidney, whereas MHC class II+ and CD163+ macrophages first appeared on neonatal days 4 and 8, respectively. Apoptotic bodies were seen in the fetal kidney and early stages of the neonatal kidney (days 1-4), and simultaneously CD68+ macrophages appeared, indicating that CD68+ macrophages may have roles in phagocytosis of apoptotic bodies and contribute to renal tissue maturation. Colony stimulating factor 1 and insulin growth factor 1 mRNAs were increased in the late stage of renal development (neonatal day 12 or later), and simultaneously CD163+ and MHC class II+ cells appeared, suggesting that these cells may be a source of these growth factors and participate in renal tissue modeling. Generally, the CD163+ and MHC class II+ cell number was much smaller than that of CD68+ cells in the developing neonatal kidney. Therefore, the obtained findings provide valuable information on the participation of macrophages in the developing rat kidney. This information may be useful for evaluation of renal toxicity when macrophages are involved in the development of renal injury.

Immunophenotypical Characterization of M1/M2 Macrophages and Lymphocytes in Cisplatin-Induced Rat Progressive Renal Fibrosis.

Renal fibrosis is regarded as the common final pathway leading to chronic kidney diseases; macrophages and myofibroblasts play important roles in the development of fibrosis. F344 rats were injected once with cisplatin (CDDP; 6 mg/kg BW) for renal lesions. Here, immunophenotypical characteristics of macrophages and lymphocytes in CDDP-induced rat renal lesions were investigated histopathologically; the CDDP-induced renal lesions consisted of tissue damage at the early-stage, worsen the damage and commencement of interstitial fibrosis at the mid-stage, and progressive fibrosis at the late stage; the KIM-1 expression and α-SMA+ myofibroblast area reflected renal tubular damage/abnormal regeneration and renal interstitial fibrosis, respectively. CD68+ M1 macrophages began to increase at the mid-stage, with increased mRNA expressions of M1-related cytokines (INF-γ, TNF-α and IL-6), and then slightly decreased at the late-stage. CD163+ M2 macrophages showed a gradually increased number at the mid- and late-stages, accompanied by increased TGF-ß1 mRNA expression (a fibrogenic factor). Double immunofluorescence using fibrotic samples at the late-stage revealed that 62.0-78.0% of CD68+ M1 macrophages co-expressed CD163, indicating that M1/M2 macrophages may contribute to progressive renal fibrosis in cooperation; further, MHC class II-expressing macrophages had a tendency towards M1 polarization, whereas CD204-expressing macrophages towards M2 polarization. In addition, CD4+ and CD8+ T cells were increased at the late-stage. Collectively, progressive renal interstitial fibrosis may be developed by complicated mechanisms that arose via interaction of M1/M2 macrophages (inflammatory for M1 and anti-inflammatory for M2) and T cells reacting to CD4 (for helper) and CD8 (for cytotoxicity). This study would provide some information on the pathogenesis of renal fibrosis based on inflammatory cells.

Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver.

The liver consists of parenchymal hepatocytes and non-parenchymal cells. Non-parenchymal cells, Kupffer cells, hepatic stellate cells and cholangiocytes have crucial roles in liver homeostasis and liver pathology. To establish baseline data, this study investigated immunohistochemically the distribution of non-parenchymal cells in perivenular areas (PV), periportal areas (PP) and Glisson's sheath (GS) of adult rat liver. Liver tissues were collected from the left lateral lobe of rats. CD163-positive macrophages were seen along the sinusoid of PV and PP areas, indicating Kupffer cells. Double immunofluorescence showed, Kupffer cells partly co-expressed CD68 and MHC class II antigens in the liver. The numbers of Kupffer cells were significantly high in PP areas as compared with PV or GS areas. CD68-positive exudative macrophages were highly localized in PP and GS areas and a comparatively low PV area. MHC class II-positive dendritic cells (activated macrophages) were localized mainly in GS. Granzyme B-positive NK cells were mainly localized in the Glisson's sheath. CD3-positive T cells and CD20-positive B cells were distributed along the sinusoids of the PP and PV areas of hepatic lobules. Vimentin and glial fibrillary acidic protein (GFAP)-positive hepatic stellate cells were localized along sinusoids in the hepatic lobules of the liver. Cholangiocytes reacting to cytokeratin 19 were seen on interlobular bile ducts in Glisson's sheath of the liver. This study shows that heterogeneous macrophage populations, liver-resident lymphocytes and hepatic stellate cells localized in PP and PV areas or GS areas of the liver with cells specific patterns.

Effect of formaldehyde and urea contaminated feed exposure into the liver of young and adult pigeons (Columba livia).

BACKGROUND AND AIM: Nowadays, toxic chemical contaminants in food are a major food safety problem in Bangladesh. Among toxic food contaminants, formalin is used to preserve fruit, vegetables, and fish, where urea is used for the whitening of rice and puffed rice. The purpose of this study was to determine the biochemical and histopathological effects on the liver of young and adult pigeons after exposure to formalin and urea contaminated feed. MATERIALS AND METHODS: A total of 15 young and 15 adult pigeons were divided into control group, formaldehyde exposed group (2.5 mL formalin/kg feed), and urea exposed (1 g/kg feed) group. Each group consisted of five pigeons. After the experimentation procedures, the blood samples were collected for biochemical study, and the liver tissue was collected for histomorphological study. The statistical analysis was performed using the Student's t-test, and p<0.05 was considered as statistically significant. RESULTS: The aspartate transaminase serum hepatic enzyme was significantly increased in both formalin and urea exposed young and adult pigeons than the control pigeons. In control pigeons, parenchymal hepatocytes and non-parenchymal cells are regularly arranged. However, histological observation of the liver of formalin and urea exposed young, and adult pigeons showed coagulation necrosis with infiltration of many inflammatory cells around the central and portal veins. The necrotic areas are more extensive with massive infiltration of inflammatory cells in the liver of formalin-treated pigeons than the urea treated pigeons. CONCLUSION: The present study results show that low concentrations of formalin and urea in feed induced liver lesions in pigeons in different extents and indicate that exposure to toxic chemicals may affect homeostasis of the liver and cause liver injury or act as a co-factor for liver disease.

Immunohistochemical characterization of myofibroblasts appearing in isoproterenol-induced rat myocardial fibrosis.

Fibrotic lesion is formed by myofibroblasts capable of producing collagens. The myofibroblasts are characterized by immunoexpressions of vimentin, desmin and α-smooth muscle actin (α-SMA) in varying degrees. The cellular characteristics remain investigated in myocardial fibrosis. We analyzed immunophenotypes of myofibroblasts appearing in isoproterenol-induced myocardial fibrosis in rats until 28 days after injection (10 mg/kg body weight); the lesions developed as interstitial edema and inflammatory cell reaction on 8 hr and days 1 and 3, and fibrosis occurred on days 1, 3, 7, 14, and 21 by gradual deposition of collagens, showing the greatest grade on day 14; the lesions gradually reduced with sporadic scar until day 28. Myofibroblasts expressing vimentin and α-SMA increased with a peak on day 3, and then, gradually decreased onwards. Interestingly, Thy-1 expressing cells appeared in the affected areas, apparently being corresponding to the grade similar to vimentin- and α-SMA-positive cells. Thy-1 is expressed in immature mesenchymal cells such as pericytes with pluripotent nature. The immunoreactivity for A3-antigen, a marker for immature mesenchymal cells, was seen in some surrounding cells. There were no cells reacting with antibodies to nestin or glial fibrillary acidic protein, although hepatic myofibroblats have been reported to react with these antibodies. Collectively, myofibroblasts appearing in rat myocardial fibrosis may have been derived from immature mesenchymal cells positive for Thy-1 or A3-antigen, with thereafter showing expressions of vimentin and α-SMA in differentiation.

Immunophenotypical analysis of pancreatic interstitial cells in the developing rat pancreas and myofibroblasts in the fibrotic pancreas in dogs and cats.

Pancreatic fibrosis develops as the results of the activity of myofibroblasts capable of producing collagens. The myofibroblasts derive from pancreatic interstitial cells, including pancreatic stellate cells (PSCs), which can express glial fibrillary acidic protein (GFAP). First, we investigated the expression patterns of vimentin, desmin, α-smooth muscle actin (α-SMA), Thy-1 and GFAP in the developing rat pancreas (in fetuses at 18 and 20 days, neonates from 1 to 21 days, and adults). Interstitial cells in the developing pancreas expressed vimentin, desmin, GFAP and Thy-1 at varying degrees; interestingly, the reactivity for desmin and vimentin was the highest in fetuses. GFAP expression was consistent between fetuses and neonates, and Thy-1 reactivity transiently increased after birth; however, α-SMA-positive interstitial cells were rarely seen. Next, we analyzed the immunophenotypical characteristics of myofibroblasts appearing in pancreatic fibrosis in dogs and cats. With increasing fibrotic grade, myofibroblasts showed increased expression of vimentin, desmin and α-SMA, in addition to increased GFAP expression. Collectively, pancreatic interstitial cells and myofibroblasts may have similar immunophenotypes, and myofibroblasts might originate partly from GFAP-expressing PSCs.

Immunolocalization of ß-catenin, E-cadherin and N-cadherin in neonate and adult rat kidney.

ß-catenin, E-cadherin and N-cadherin are adhesion molecules that play important roles in organogenesis, tissue homeostasis, renal epithelial integrity and polarity. The present study demonstrated their immunolocalization in adult and neonate rat kidney. Membranous or cytoplasmic expression of ß-catenin, E-cadherin and N-cadherin were seen in adult and developing renal tubular epithelial cells. Particularly, in adult kidney, E-cadherin and ß-catenin were intensively expressed in distal renal tubules, whereas N-cadherin was expressed in proximal renal tubules. In neonate rat kidney on 1 and 4 days old, developing renal tubular epithelial cells were mainly reacted with E-cadherin and very weakly expressed N-cadherin; ß-catenin was expressed in developing renal tubules and mesenchymal blastemal cells. Interestingly, ß-catenin-positive renal tubular epithelial cells simultaneously expressed E-cadherin in the kidney of adult and developing rats. Collectively, the adhesion molecules were differentially distributed in the renal tubules of adult rats and ß-catenin and E-cadherin are predominant adhesion molecules in developing kidney. The present findings would provide the basic information of evaluating renal tubular toxicity using rats, in addition to renal genesis, in terms of adhesion molecules.

Glutamatergic thalamopallial projections in the pigeon identified by retrograde labeling and expression of vGluT2 mRNA.

A neocortical hypothesis as to homology of certain nuclear components of the avian brain proposes that the entopallium and field L2 are homologous to layer 4 of mammalian extrastriate and auditory neocortex, respectively. However, the hypothesis lacks support from the neurochemistry of thalamopallial projections. We investigated whether these projections are glutamatergic by injecting cholera toxin B into either the entopallium or field L2 in combination with in situ hybridization. Retrogradely labeled neurons in nucleus rotundus and nucleus ovoidalis were found to express vesicular glutamate transporter 2 mRNA, showing that the thalamopallial projections are glutamatergic. The results are consistent with the neocortical hypothesis.

Expression of vesicular glutamate transporter 3 mRNA in the brain and retina of the pigeon.

Vesicular glutamate transporters (vGluTs), which accumulate glutamate into synaptic vesicles, are classified into three subtypes in mammalian brains: vGluT1, vGluT2, and vGluT3. VGluT3 is localized in non-glutamatergic neurons of the brain and retinal amacrine cells. In birds, the vGluT3 genome is found, but its distribution in the brain or retina is unknown. The present study was conducted to analyze vGluT3 cDNA sequence and elucidate its distribution in the pigeon brain and retina. The vGluT3 cDNA comprises 1761bp and showed 95% and 88% identity to the chicken and zebra finch vGluT3 cDNAs, respectively, and 74% identity to human vGluT3 cDNA. In situ hybridization revealed that the vGluT3 mRNA was expressed in neurons of the caudal linear nucleus (LC) of the brain and in amacrine cells of the inner nuclear layer of the retina. A combination of in situ hybridization and serotonin immunohistochemistry revealed three types of stained cells in LC and retina: vGluT3(+)/serotonin(+), vGluT3(+)/serotonin(-), and vGluT3(-)/serotonin(+). The vGluT3(+)/serotonin(+) cells were approximately 22% in LC and 16% in the retina. The present results suggest that the pigeon vGluT3 mRNA is comparable with the mammalian type.

Homology of the mesopallium in the adult chicken identified by gene expression of the neocortical marker cholecystokinin.

Studies of gene expression and fiber connections have suggested that the primary visual (entopallium) and auditory (field L2) centers in the avian telencephalon are homologous to layer 4 of extrastriate and auditory neocortices of mammals, respectively. In addition, it has been proposed that the arcopallium contains neurons homologous to layers 5/6 and that the mesopallium may be homologous to superficial neocortical layers, but gene expression evidence for the latter is lacking in adult birds. In the present study using adult chickens we have examined the gene expression of cholecystokinin (CCK) mRNA, a selective marker for layers 2/3 of mammalian neocortex. CCK mRNA was expressed in neurons of the entire mesopallium, but not in any part of the nidopallium. Together with hodological evidence of connections between the mesopallium and the two primary sensory areas, our results are consistent with the suggestion that the mesopallium is comparable to certain superficial layers of mammalian neocortex.

Distribution of vesicular glutamate transporter 2 in auditory and song control brain regions in the adult zebra finch (Taeniopygia guttata).

The songbird brain has a system of interconnected nuclei that are specialized for singing and song learning. Wada et al. (2004; J. Comp. Neurol. 476:44-64) found a unique distribution of the mRNAs for glutamate receptor subunits in the song control brain areas of songbirds. In conjunction with data from electrophysiological studies, these finding indicate a role for the glutamatergic neurons and circuits in the song system. This study examines vesicular glutamate transporter 2 (VGLUT2) mRNA and protein expression in the zebra finch brain, particularly in auditory areas and song nuclei. In situ hybridization assays for VGLUT2 mRNA revealed high levels of expression in the ascending auditory nuclei (magnocellular, angular, and laminar nuclei; dorsal part of the lateral mesencephalic nucleus; ovoidal nucleus), high or moderate levels of expression in the telencephalic auditory areas (cudomedial mesopallium, field L, caudomedial nidopallium), and expression in the song nuclei (HVC, lateral magnocellular nucleus of the anterior nidopallium, robust nucleus of the arcopallium), where levels of expression were greater than in the surrounding brain subdivisions. Area X did not show expression of VGLUT2 mRNA. Nuclei in the descending motor pathway (dorsomedial nucleus of the intercollicular complex, retroambigual nucleus, tracheosyringeal motor nucleus of the hypoglossal nerve) expressed VGLUT2 mRNA. The target nuclei of VGLUT2 mRNA-expressing nuclei showed immunoreactivity for VGLUT2 as well as hybridization signals for the mRNA of glutamate receptor subunits. The present findings demonstrate the origins and targets of glutamatergic neurons and indicate a central role for glutamatergic circuits in the auditory and song systems in songbirds.

Expression of the neocortical marker, RORß, in the entopallium and field L2 of adult chicken.

Two opposing hypotheses on the homology of the avian brain suggest that the dorsal ventricular ridge of birds is comparable in certain respects either to the neocortex or to the claustroamygdalar complex of mammals. To help resolve this issue, we examined in adult chicken brains the gene expression of RORß mRNA, a selective marker for layer IV of mammalian neocortex. RORß mRNA was expressed in neurons of the chicken's visual entopallium and auditory field L2, but not in other regions of the nidopallium, hyperpallium, mesopallium or arcopallium. Together with hodological evidence of direct thalamic projections conveying primary sensory information to the entopallium and field L2, our results support the contention that these two regions are composed of neurons comparable to those in layer IV of mammalian neocortex.