Difference in the Mechanism of Iron Overload-Enhanced Acute Hepatotoxicity Induced by Thioacetamide and Carbon Tetrachloride in Rats.

Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.

Myelin lesion in the aspartoacylase (Aspa) knockout rat, an animal model for Canavan disease.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Functional role of IL-19 in a mouse model of L-arginine-induced pancreatitis and related lung injury.

IL-19 is a member of IL-10 family and is mainly produced by macrophages. Acute pancreatitis (AP) is an inflammatory disease characterized by acinar cell injury and necrosis. In the present study, the role of IL-19 in AP and AP-associated lung injury in mice was explored using L-arginine-induced pancreatitis. Experimental pancreatitis was induced by intraperitoneal injection of L-arginine in wild-type (WT) and IL-19 gene deficient (IL-19 KO) mice. In L-arginine treated mice, the serum amylase level was significantly increased in IL-19 KO mice, and interstitial edema, analyzed using hematoxylin and eosin (H&E)-stained sections, was aggravated mildly in IL-19 KO mice compared to WT mice. Compared to WT mice treated with L-arginine, mRNA expression of tumor necrosis factor (TNF)-α was significantly upregulated in IL-19 KO mice treated with L-arginine. In WT mice, IL-19 mRNA was equally expressed in the pancreas of both control and L-arginine treated mice. The condition of lung alveoli in WT and IL-19 KO mice treated with L-arginine was then evaluated. In mice with L-arginine-induced pancreatitis, alveolar area was remarkedly decreased, and expression of lung myeloperoxidase was significantly increased in IL-19 KO mice compared to WT mice. In the lungs, mRNA expressions of IL-6 and inducible nitric oxide synthase were significantly increased in IL-19 KO mice compared to WT mice. In summary, IL-19 was proposed to alleviate L-arginine-induced pancreatitis by regulating TNF-α production and to protect against AP-related lung injury by inhibiting neutrophil migration.

Pleural mesothelioma in a California sea lion (Zalophus californianus).

A 25-year-old female California sea lion (Zalophus californianus) reared in an aquarium died following a history of anorexia, lethargy, abnormal protrusion of the skin, and oral respiration. At necropsy, multiple yellowish-white nodules with diameters of 0.1-0.5 cm were disseminated in the thoracic cavity and lungs. Histopathologically, the nodules were continuous with normal mesothelium and were characterized by the proliferation of spindle-shaped to polygonal neoplastic cells with prominent atypia. The neoplastic cells exhibited diffuse, strong staining for vimentin and partial, weak to moderate staining for cytokeratin AE1/AE3. Based on these findings, the lesions were diagnosed as pleural mesothelioma. This study reports the first case of pleural mesothelioma in California sea lion.

Analyses of damage-associated molecular patterns, particularly biglycan, in cisplatin-induced rat progressive renal fibrosis.

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan.

Successful management of nasopharyngitis caused by Schizophyllum commune in a captive cheetah (Acinonyx jubatus).

Herein, we describe the management of nasopharyngitis caused by Schizophyllum commune infection in a captive cheetah. Computed tomography revealed a nodule in the nasal cavity and pharynx, and an endoscopic biopsy was performed. As a result, the nodule was surgically resected because of a suspected carcinoma. However, the surgical specimen was histologically re-evaluated and a fungal granuloma was diagnosed. Sequence analysis of DNA from formalin-fixed, paraffin-embedded samples revealed S. commune infection. The cheetah was administered fluconazole orally for 73 days. However, the drug was ineffective and itraconazole was administered for 14 days. Symptoms such as nasal discharge and sneezing have completely resolved for 4 years.

Interleukin-19 Gene-Deficient Mice Promote Liver Fibrosis via Enhanced TGF-ß Signaling, and the Interleukin-19-CCL2 Axis Is Important in the Direction of Liver Fibrosis.

IL-19 is a cytokine discovered by homologous searching with IL-10 and is produced by non-immune cells, such as keratinocytes, in addition to immune cells, such as macrophages. Liver fibrosis results from the inflammation and activation of hepatic stellate cells via chronic liver injury. However, the participation of IL-19 in liver fibrosis remains to be sufficiently elucidated. Our group studied the immunological function of IL-19 in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. IL-19 gene-deficient (KO) mice and body weight-matched wild-type (WT) mice were used. A liver fibrosis mouse model was created via CCl4 administration (two times per week) for 8 weeks. In CCl4-induced liver fibrosis, serum analysis revealed that IL-19 KO mice had higher ALT levels compared to WT mice. IL-19 KO mice had worse fibrosis, as assessed by morphological evaluation of total area stained positive with Azan and Masson trichrome. In addition, the expression of α-SMA was increased in liver tissues of IL-19 KO mice compared to WT mice. Furthermore, mRNA expression levels of TGF-ß and α-SMA were enhanced in IL-19 KO mice compared to WT mice. In vitro assays revealed that IL-19-high expressing RAW264.7 cells inhibited the migration of NIH3T3 cells via the inhibited expression of CCL2 in the presence of CCl4 and IL-4. These findings indicate that IL-19 plays a critical role in liver fibrosis by affecting TGF-ß signaling and the migration of hepatic stellate cells during liver injury. Enhancement of the IL-19 signaling pathway is a potential treatment for liver fibrosis.

Disrupted neurogenesis, gliogenesis, and ependymogenesis in the Ccdc85c knockout rat for hydrocephalus model.

Coil-coiled domain containing 85c (Ccdc85c) is a causative gene for congenital hydrocephalus and subcortical heterotopia with frequent brain hemorrhage. We established Ccdc85c knockout (KO) rats and investigated the roles of CCDC85C and intermediate filament protein expression, including nestin, vimentin, GFAP, and cytokeratin AE1/AE3 during the lateral ventricle development in KO rats to evaluate the role of this gene. We found altered and ectopic expression of nestin and vimentin positive cells in the wall of the dorso-lateral ventricle in the KO rats during development from the age of postnatal day (P) 6, whereas both protein expression became faint in the wild-type rats. In the KO rats, there was a loss of cytokeratin expression on the surface of the dorso-lateral ventricle with ectopic expression and maldevelopment of ependymal cells. Our data also revealed disturbed GFAP expression at postnatal ages. These findings indicate that lack of CCDC85C disrupts the proper expression of intermediate filament proteins (nestin, vimentin, GFAP, and cytokeratin), and CCDC85C is necessary for normal neurogenesis, gliogenesis, and ependymogenesis.

The effect of lipopolysaccharide on liver homeostasis and diseases based on the mutual interaction of macrophages, autophagy, and damage-associated molecular patterns in male F344/DuCrlCrlj rats.

Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.

L-arginine-induced pancreatitis aggravated by inhibiting Na+/Ca2+ exchanger 1.

Na+/Ca2+ exchangers (NCX) are an exchange transporter of Na+ and Ca2+ ions on the plasma membrane. There are three types of NCX: NCX1, NCX2, and NCX3. We have been working for many years to understand the role of NCX1 and NCX2 in gastrointestinal motility. In this study, we focused on the pancreas, an organ closely related to the gastrointestinal tract, and used a mouse model of acute pancreatitis to investigate a possible role for NCX1 in the pathogenesis of pancreatitis. We characterized a model of acute pancreatitis induced by excessive doses of L-arginine. We administered the NCX1 inhibitor SEA0400 (1 mg/kg) 1 hr prior to L-arginine-induced pancreatitis and evaluated pathological changes. Mice treated with NCX1 inhibitors show exacerbation of the disease with decreased survival and increased amylase activity in response to L-arginine-induced experimental acute pancreatitis, and this exacerbation correlates with increased autophagy mediated by LC3B and p62. These results suggest that NCX1 has a role in regulating pancreatic inflammation and acinar cell homeostasis.

Macrophage pathology in hepatotoxicity.

The liver is the most important organ that metabolizes and detoxifies chemicals taken into the body. Therefore, there is always a risk of liver damage owing to the toxic effects of chemicals. The mechanisms of hepatotoxicity have been studied extensively and deeply based on toxic effects of chemicals themselves. However, it is important to note that liver damage is variously modified by the patho-biological reactions evoked mainly via macrophages. Macrophages appearing in hepatotoxicity are evaluated by the M1/M2 polarization; M1 macrophages promote tissue injury/inflammation, whereas M2 macrophages show anti-inflammatory action including reparative fibrosis. The "portal vein-liver barrier" regulated by Kupffer cells and dendritic cells in and around the Glisson's sheath may be related to the initiation of hepatotoxicity. In addition, Kupffer cells exhibit the two-sides of functions (that is, M1 or M2 macrophage-like functions), depending on microenvironmental conditions which may be raised in part by gut microbiota-derived lipopolysaccharide. Furthermore, damage-associated molecular patterns (DAMPs) (in particular, HMGB1) and autophagy (which degrades DAMPs) also play roles in the polarity of M1/M2 macrophages. The mutual relation of "DAMPs (HMGB-1)-autophagy-M1/M2 macrophage polarization" as the patho-biological reaction should be taken into consideration in hepatotoxicity evaluation.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

Dietary iron overload enhances Western diet induced hepatic inflammation and alters lipid metabolism in rats sharing similarity with human DIOS.

Hepatic iron overload is often concurrent with nonalcoholic fatty liver disease (NAFLD). Dysmetabolic iron overload syndrome (DIOS) is characterized by an increase in the liver and body iron stores and metabolic syndrome components. Increasing evidences suggest an overlap between NAFLD with iron overload and DIOS; however, the mechanism how iron is involved in their pathogenesis remains unclear. Here we investigated the role of iron in the pathology of a rat model of NAFLD with iron overload. Rats fed a Western (high-fat and high-fructose) diet for 26 weeks represented hepatic steatosis with an increased body weight and dyslipidemia. Addition of dietary iron overload to the Western diet feeding further increased serum triglyceride and cholesterol, and enhanced hepatic inflammation; the affected liver had intense iron deposition in the sinusoidal macrophages/Kupffer cells, associated with nuclear translocation of NFκB and upregulation of Th1/M1-related cytokines. The present model would be useful to investigate the mechanism underlying the development and progression of NAFLD as well as DIOS, and to elucidate an important role of iron as one of the "multiple hits" factors.

Spinal epidural empyema concurrent with sequestrum in a cat: a case report.

A 5-month-old intact female mixed cat presented with repetitive paraplegia and drainage of pus from the back despite continuous antibiotic medication. Neurologic examination was consistent with below T3-L3 myelopathy. Computed tomography and magnetic resonance imaging revealed a contrast-enhanced mass in the L1-3 spinal canal, and bone fragments in the T13 and L1 spinal canal. Spinal epidural empyema was suspected, and hemilaminectomy was performed for T12-L2 on the right side and T11-12 on the left side. Bone fragments were diagnosed as sequestrum infected with Bacteroides sp. The cat recovered enough to ambulate next day. One month after surgery, there was no deficit in neurological function. This is the first report of spinal epidural empyema concurrent with sequestrum in a cat.

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.

Expression of CCDC85C, a causative protein for hydrocephalus, and intermediate filament proteins during lateral ventricle development in rats.

Coiled-coil domain containing 85c (Ccdc85c) is a causative gene for genetic hydrocephalus and subcortical heterotopia with frequent brain hemorrhage. In the present study, we examined the expression pattern of CCDC85C protein and intermediate filament proteins, such as nestin, vimentin, GFAP, and cytokeratin AE1/AE3, during lateral ventricle development in rats. CCDC85C was expressed in the neuroepithelial cells of the dorsal lateral ventricle wall, diminishing with development and almost disappearing at postnatal day 20. By immunoelectron microscopy, CCDC85C was localized in the cell-cell junction and apical membrane. The expression of nestin and vimentin was decreased in the wall of the lateral ventricle in manner similar to CCDC85C, but GFAP expression started immediately after birth and became stronger with age. Moreover, cytokeratin expression was found at postnatal day 13 and increased at postnatal day 20 in conjunction with the disappearance of CCDC85C expression. Taken together, CCDC85C is expressed in the cell-cell junctions lining the wall of the lateral ventricle and plays a role in neural development with other intermediate filaments in the embryonic and postnatal periods. Our chronological study will help to relate CCDC85C protein with intermediate filaments to elucidate the detailed role of CCDC85C protein during neurogenesis.

IL-19 Contributes to the Development of Nonalcoholic Steatohepatitis by Altering Lipid Metabolism.

Interleukin (IL)-19, a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. Nonalcoholic steatohepatitis (NASH) is a disease that has progressed from nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. We evaluated the functions of IL-19 in a NAFLD/NASH mouse model using a 60% high fat diet with 0.1% methionine, without choline, and with 2% cholesterol (CDAHFD). Wild-type (WT) and IL-19 gene-deficient (KO) mice were fed a CDAHFD or standard diet for 9 weeks. Liver injury, inflammation, and fibrosis induced by CDAHFD were significantly worse in IL-19 KO mice than in WT mice. IL-6, TNF-α, and TGF-ß were significantly higher in IL-19 KO mice than in WT mice. As a mechanism using an in vitro experiment, palmitate-induced triglyceride and cholesterol contents were decreased by the addition of IL-19 in HepG2 cells. Furthermore, addition of IL-19 decreased the expression of fatty acid synthesis-related enzymes and increased ATP content in HepG2 cells. The action of IL-19 in vitro suppressed lipid metabolism. In conclusion, IL-19 may play an important role in the development of steatosis and fibrosis by directly regulating liver metabolism and may be a potential target for the treatment of liver diseases.