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.

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.

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.

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.

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.

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.

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.

Systemic Anaplastic Large T-Cell Lymphoma with Initial Presentation of Dysuria in a Dog.

A 6-year-old spayed female Akita Dog had dysuria, severe urinary retention and miliary masses in the vagina. Computed tomography revealed a mass at the urethrovaginal junction. The dog died 2 months after initial presentation. At necropsy, the urethrovaginal mass was greyish‒white, solid and 9 × 6 × 6 cm in size with circumferential thickening of the urethral wall. Multiple whitish nodules were seen in the visceral organs and skin. Histopathologically, the urethrovaginal mass comprised a diffuse population of medium-sized to large round neoplastic cells with ovoid to bean-shaped nuclei and eosinophilic cytoplasm. Aberrantly large neoplastic cells with eccentric, horseshoe-shaped or irregularly-shaped nuclei and abundant eosinophilic cytoplasm resembled 'hallmark cells' of human anaplastic large cell lymphoma. Similar neoplastic lesions were present in all the grossly visible masses. Neoplastic cells were diffusely immunopositive for CD3 and occasionally for CD30 and granzyme B. On the basis of the clinical, pathological and immunohistochemical findings, the case was diagnosed as systemic anaplastic large cell lymphoma arising from the lower urinary tract.

Radiotherapy-induced tumor lysis syndrome in a dog with thymoma.

A 13-year-old, female, mixed-breed dog with a huge cranial mediastinal mass underwent radiotherapy (RT). On the following day, the dog presented with lethargy and anorexia. Hematological examination revealed elevated levels of blood urea nitrogen, creatinine, inorganic phosphorus, potassium, lactate dehydrogenase, creatine phosphokinase and aspartate aminotransferase, decreased calcium level, and metabolic acidosis. Urine output markedly decreased. The patient recovered with fluid therapy and diuretic therapy; however, died suddenly from an unknown cause 11 days after RT completion. Histopathological examination after necropsy showed thymoma in the cranial mediastinum and extensive tubular necrosis of both kidneys which may be due to RT-induced tumor lysis syndrome (TLS). This report suggests that the risk of TLS should be evaluated in dogs with thymoma who undergo RT.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

PHF24 is expressed in the inhibitory interneurons in rats.

Noda epileptic rat (NER) is a mutant model for epilepsy that exhibits spontaneous generalized tonic-clonic seizure. Epileptogenesis of NER remains to be elucidated; but it is detected an insertion of an endogenous retrovirus sequence in intron 2 of the PHD finger protein 24 (Phf24) gene, encoding Gαi-interacting protein (GINIP). Phf24 is a strong candidate gene for epileptogenesis in NER. PHF24 modulates GABAB signaling through interacting with Gαi protein. To clarify the epileptogenesis of NER, we investigated a distribution of PHF24-expressing cells in the central nerve system (CNS). While broad expression of PHF24 was observed in the CNS, characteristic expression was noted in the periglomerular layer of the olfactory bulb and the lamina II of the spinal cord in the control rats. These cells showed co-expression with calbindin or calretinin, inhibitory interneuron markers. In the olfactory bulb, 15.6% and 41.2% of PHF24-positive neurons co-expressed calbindin and calretinin, respectively. Immunoelectron microscopy revealed that PHF24 was located in the presynaptic terminals, synaptic membranes and cytoplasmic matrix of neuronal soma. Our data suggested PHF24 is expressed in the inhibitory interneurons and may play important roles in modulation of the GABAB signaling.

Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

Assessment of biomarkers influencing treatment success on small intestinal lymphoma in dogs.

This study aimed to determine a reliable therapeutic biomarker for localized small intestinal lymphoma (SIL) in dogs based on clinical and histopathological features. We retrospectively investigated 84 dogs with localized SIL, including 36 dogs receiving surgery and 48 dogs receiving chemotherapy. The dogs receiving surgery were divided into two subgroups: 18 dogs (group 1) with overall survival (OS) <120 days (median OS) and 18 dogs (group 2) with OS ≥120 days. Correspondingly, the dogs receiving chemotherapy were divided into 24 dogs (group 3) with OS <98 days (median OS) and 24 dogs (group 4) with OS ≥98 days. Clinical, haematological, histopathological and immunohistochemical analyses were comparatively evaluated among the four subgroups. There was no significant difference in OS between the surgery and chemotherapy groups. In dogs receiving surgery, the rate of Ki67-positive cells was significantly increased in group 1 compared to group 2 and showed no significant difference between groups 3 and 4. In dogs receiving chemotherapy, the rate of O6-methylguanine-DNA methyltransferase (MGMT) was significantly higher in group 3 than in group 4 and showed no significant difference between groups 1 and 2. Additionally, our data showed that OS in dogs with higher Ki67 expression might be significantly increased by chemotherapy than by surgery, that of those with higher MGMT expression might be significantly increased by surgery than by chemotherapy, and Ki67 and MGMT were independent of each other. Indices of Ki67 and MGMT are suggested therapeutic biomarkers to determine the optimal first-line treatment for localized SIL in dogs.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Participation of Somatic Stem Cells, Labeled by a Unique Antibody (A3) Recognizing both N-glycan and Peptide, to Hair Follicle Cycle and Cutaneous Wound Healing in Rats.

A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues.