Pathological analysis of lesions in the exocrine pancreas of rats induced by Zinc Maltol.

The pancreas plays an important role in the homeostasis of zinc (Zn), a nutritionally essential metal. In several previous studies, Zn ions induced inflammatory changes in the exocrine pancreas; however, little is known about Zn complexes. In this study, we microscopically, immunohistochemically, and ultrastructurally examined pancreatic lesions in Sprague-Dawley (SD) rats induced by a 4-week repeated oral dose toxicity study of Zinc Maltol (ZM), a zinc (II) complex. ZM induces acinar atrophy and increases the number of duct-like structures. Immunohistochemistry revealed a decrease in the number of trypsin-positive cells, and an increase in the number of SOX9-positive cells. Interstitial fibrosis and macrophage infiltration also correlated with the degree of acinar atrophy. Electron microscopic evaluation revealed that the acinar cells that lost granules were surrounded by fibroblasts and collagen fibers. In conclusion, we provided a detailed description of ZM-induced pancreatic lesions in SD rats.

Malignant stromal cell tumor of the spleen in a WBN/Kob rat.

Primary splenic stromal tumors have rarely been reported in rodents. We report the case of a 90-week-old male WBN/Kob rat with a nodular demarcated mass in the spleen, which was kept as a non-treated animal in a long-term animal study. Histopathology revealed round to short spindle-shaped tumor cells arranged in a solid growth pattern. Invasive growth, anisokaryosis, and high mitotic activity (46 per 10 high-power fields [2.37 mm2]) were observed to be multifocal, but most tumor cells showed mild nuclear pleomorphism. The pattern of silver impregnation corresponded to that of the marginal zone of the red pulp. Immunohistochemistry revealed that the tumor cells were double positive for fascin and desmin and focally positive for Iba-1 and OX-6 expression. These characteristics were similar to those observed in fibroblastic reticular cells and dendritic cells in the marginal zone of the red pulp. These findings suggest that the malignant stromal cell tumor of the spleen in this case had characteristics of both fibroblastic reticular cells and dendritic cells.

Comparative study on detectability of learning and memory disorder between two water maze tests commonly used in juvenile rat toxicity studies using isoflurane inhaled rat model.

Evaluation of learning and memory is crucial in juvenile animal toxicity studies (JAS) during the development of CNS active drugs, but there are no currently recommended test methods. We compared the ability of the Morris water maze (MWM) and the Biel water maze (BWM) to detect learning and memory disorder (LMD) using rats inhaled isoflurane (IFN). Rats were treated with 1% IFN using inhalation on postnatal day (PND) 7 for 6 h. All rats were subjected to the MWM on PND 33 and the BWM on PND 55/57 (Experiment 1), or the BWM on PND 32/33 and the MWM on PND 54/55 (Experiment 2). On PND 70, the brain was weighed and then neurohistopathology was conducted. There were no IFN-related changes in clinical signs and body weight. In the tests beginning on PND 32/33, the MWM clearly detected IFN-related LMD in both sexes whereas the BWM detected LMD only in males. With an additional benefit of a simpler procedure, the MWM was considered superior to the BMW for JAS. LMD was not detected in both mazes tested from PND 54/55/57, which was considered due to weak effect and/or recovery of brain function with growth. Single IFN inhalation on PND 7 was considered useful as positive control to induce LMD caused by postnatal exposure in rats, but stronger treatment regimens was recommended.

High-fat diet-induced nonalcoholic steatohepatitis is accelerated by low carnitine and impaired glucose tolerance in novel murine models.

Carnitine deficiency and impaired glucose tolerance (IGT) exacerbate liver steatosis. Given the current lack of ideal murine nonalcoholic steatohepatitis (NASH) models, we investigated new NASH models using jvs/+ mice with low carnitine and wild-type mice with low-dose alloxan-induced IGT. The jvs/+ and wild-type mice were divided into jvs/+ mice fed a high-fat diet (HFD) from 3 weeks of age (HF hetero group), wild-type mice with low-dose alloxan treatment fed HFD (AL + HF wild group), wild-type mice fed HFD (HF wild group), and two types of mice fed a normal diet-jvs/+ and wild-type (intact group). All mice were sacrificed at 20 or 40 weeks of age. All male HFD-fed mice showed obesity, IGT, high blood insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR), high liver enzyme levels, and high cholesterol levels. The degree of IGT was the worst in the AL + HF wild group, and blood insulin levels and HOMA-IR score were remarkably increased from 20 to 40 weeks of age. Almost all HFD-fed mice showed steatosis, fibrosis, and lobular inflammation in the centrilobular zone. These changes were accompanied by hepatocyte ballooning and were enhanced at 40 weeks of age. Furthermore, the incidence rate of nodular hyperplasia and adenoma in both the HF hetero and AL + HF wild groups was nearly 30%. We successfully established two novel murine models of NASH using male jvs/+ mice with low carnitine and male wild-type mice with IGT that eventually developed obesity, fatty liver, insulin resistance, liver fibrosis, and tumorigenesis. These results suggest that low carnitine levels and early-stage induction of IGT are important factors in the progression of NASH to tumorigenesis, similar to human NASH.

Spontaneous cholangiofibrosis adjacent to a dilated common bile duct with intestinal metaplasia in a Royal College of Surgeons rat.

A 130-week-old male Royal College of Surgeons rat kept as a non-treated animal in a long-term animal study presented with a mass in the hepatic portal region that adhered to a dilated common bile duct and the duodenum. Histopathologically, the solitary mass showed expansive growth with no apparent compression and continued to dilate the common bile duct, which had a hyperplastic epithelium with intestinal metaplasia. The mass mainly consisted of small to large dilated and/or tortuous ducts with abundant dense connective tissue and many inflammatory cells. The single-layer lining epithelium of the duct changed from cuboidal to columnar. Immunohistochemically, the lining cells were positive for cytokeratin 7, cytokeratin 19, and OV-6, which are bile duct markers. Based on the pathological characteristics, the rat was diagnosed as spontaneous cholangiofibrosis adjacent to a dilated common bile duct with intestinal metaplasia.

Hyperglycemia Suppresses Age-Related Increases in Corneal Peripheral Sensory Nerves in Wistar Bon Kobori (WBN/Kob) Rats.

Purpose: Nerve fiber density in the cornea is an alternative marker for diabetic peripheral neuropathy combined with intraepidermal nerve fiber density (IENFD). Recent studies investigated corneal nerves using rodent models of diabetes. Male Wistar Bon Kobori (WBN/Kob) rats spontaneously develop long-lasting diabetes and human-like diabetic peripheral neuropathy with vascular lesions. This study investigated corneal nerve fiber density and IENFD in diabetic male WBN/Kob rats as morphological markers of diabetic peripheral neuropathy. Methods: Male WBN/Kob rats exhibit abnormal glucose tolerance and diabetes at approximately 30 weeks of age, which progresses until approximately 90 weeks of age. Male WBN/Kob rats aged 36 and 90 weeks were therefore used for histological investigations and compared with age-matched nondiabetic female rats. Results: Terminal epithelial nerve density and subbasal nerve plexus density in the central cornea were significantly greater in nondiabetic female rats aged 90 weeks when compared with nondiabetic female rats aged 36 weeks. However, terminal epithelial nerve density and subbasal nerve plexus density did not increase with age in diabetic male WBN/Kob rats, instead lowering by up to 40%, relative to measurements in nondiabetic female rats aged 90 weeks. However, this difference was not statistically significant. IENFD was significantly lower in diabetic male rats aged 90 weeks than in male rats aged 36 weeks, but did not differ between diabetic male rats and nondiabetic female rats aged 90 weeks. Conclusions: In WBN/Kob rats, hyperglycemia suppresses an age-related increase in peripheral sensory corneal nerve density; therefore, corneal sensory nerves may be important morphological markers of diabetic peripheral sensory neuropathy.

Extended Duration of Hyperglycemia Result in Human-Like Corneal Nerve Lesions in Mice With Alloxan- and Streptozotocin-Induced Type 1 Diabetes.

Purpose: Previous experimental studies assessing corneal nerves as a measure of the severity of diabetic peripheral neuropathy have yielded discordant results; this may have been due to the effect of the short duration of the induced diabetes. We investigated whether increases in the duration of hyperglycemia result in the development of corneal lesions in a mouse model of alloxan (AL)- or streptozotocin (STZ)-induced type 1 diabetes. We further determined whether corneal nerve fiber density, intraepidermal nerve fiber density (IENFD), and sural nerve morphology can be used as morphologic markers of diabetic peripheral neuropathy in rodent models. Methods: A total of 30 female ICR mice were divided into three groups: those with STZ-induced (STZ group) and AL-induced (AL group) diabetes, and a control group. Hyperglycemia was maintained in diabetic mice for 35 weeks. Animals were euthanized at 41 weeks of age. Results: Subbasal nerve plexus density (SBNPD) and terminal epithelial nerve density (TEND) in the cornea, as well as IENFD, were significantly lower, and mean sural nerve axon sizes were smaller in mice in the STZ and AL groups than in the control group. There were significant correlations between IENFD and SBNPD, and between IENFD and TEND. Conclusions: These results indicate that the TEND and SBNTD of the cornea may be useful morphologic markers for diabetic peripheral neuropathy.

Effect of combined dyslipidemia and hyperglycemia on diabetic peripheral neuropathy in alloxan-induced diabetic WBN/Kob rats.

Clinical and experimental research have suggested that dyslipidemia aggravates diabetic peripheral neuropathy (DPN). However, whether dyslipidemia is a risk factor for DPN remains unclear. To investigate the effect of dyslipidemia on DPN, morphological features of peripheral nerves were analyzed in diabetic rats treated with a high-fat diet (HFD). Male rats were divided into four groups: nondiabetic rats (N), alloxan-induced diabetic rats (AL), diabetic rats treated with an HFD (AH), and nondiabetic rats treated with an HFD (HF). Combined hyperglycemia and dyslipidemia (AH group) induced a significant increase in plasma triglyceride and cholesterol levels. In addition, the combined effects contributed to a reduction in myelin size and a reduction in myelin thickness as indicated on sensory sural nerve histograms. There was also a reduction in the size of motor nerve axons when compared with the effects of hyperglycemia or dyslipidemia alone. However, the sensory nerve conduction velocity in the AH group was slightly but not significantly lower than those in the HF and AL groups. These results suggest that combined hyperglycemia and dyslipidemia induced mild peripheral motor and sensory nerve lesions, without significantly affecting sensory nerve conduction velocity.

Induction of Severe Chronic Hyperplastic Candidiasis in Rat by Opportunistic Infection of C. albicans through Combination of Diabetes and Intermittent Prednisolone Administration.

Chronic hyperplastic candidiasis progresses from squamous cell hyperplasia to squamous cell carcinoma (SCC); however, the oncogenic mechanism remains unclear. In the present study, we attempted to induce opportunistic Candida albicans infection and establish chronic hyperplastic candidiasis in rats by combining diabetic condition and prednisolone administration, followed by analysis of the inflammatory cells involved in the disease progression. Female Wistar Bunn/Kobori (WBN/Kob) rats were divided into 3 groups: alloxan-induced diabetic rats (A group) along with diabetic (AP group) and nondiabetic (P group) rats intermittently treated with prednisolone. Animals were euthanized at 42 weeks of age. Squamous cell hyperplasia following C. albicans infection in the forestomach was observed in almost all AP and A group rats. The lesions in the AP group were significantly more severe than those in the A group. In addition, SCC was detected in 1 AP group animal. Cluster of differentiation (CD)4-positive T cell and CD68-positive macrophage infiltration in the AP group was significantly stronger than that in the A group. These findings suggest that the combination of diabetes and intermittent prednisolone administration could induce chronic hyperplastic candidiasis without direct C. albicans inoculation and that CD4-positive T cells and CD68-positive macrophages may be highly involved in the pathogenesis of these hyperplastic lesions.

Acute alloxan renal toxicity in the rat initially causes degeneration of thick ascending limbs of Henle.

Alloxan (AL) is a material well-known to induce diabetes. Prior to inducing a prolonged diabetic state, AL causes acute tubulointerstitial nephritis. However, the precise primary target site and mechanism of its nephrotoxicity remain unclear. The objective of this study was to evaluate the morphological characteristics relevant to acute renal toxicity following AL administration. Rats were intravenously treated with AL. Eight hours after AL treatment, aquaporin 1-negative and Na/K pump-positive thick ascending limbs of Henle (TAL) were degenerated in the outer medulla. These tubular lesions progressed from the outer medulla to the cortex. At day 2 after AL treatment, the lesions reached a peak, then both proximal and distal tubules also showed degeneration and necrosis, and tubular regeneration was seen in TAL. Immunohistochemically, damaged tubular epithelium included slightly enlarged prohibitin-positive granules, but it expressed no GLUT2, which is an AL transporter. Ultrastructurally, cytoplasmic and mitochondrial swelling was detected in degenerated cells of TAL. These findings suggest that AL initially causes degeneration of TAL, and induces mitochondrial and cellular damage in the tubular epithelium without involving GLUT2.

Malignant mast cell tumor of the thymus in an Royal College of Surgeons (RCS) rat.

A 152-week-old male Royal College of Surgeons (RCS) rat kept as a non-treated animal in a long-term animal study presented with a soft mass in the anterior mediastinum, which adhered to the pleura of the lung. Histopathologically, the mass mainly consisted of round to short spindle-shaped tumor cells that had infiltrated through the hyperplastic thymic tissue. The tumor cells were arranged in loose to dense sheets. Nuclei were moderate in size and round to spindle-shaped, with small nucleoli. Almost all tumor cells exhibited abundant eosinophilic cytoplasm, including eosinophilic granules of a range of sizes. The granules of tumor cells exhibited metachromasia with toluidine blue stain and were positive for c-kit and mast cell protease II. These findings indicate that the tumor described here represents a rare case of spontaneous malignant mast cell tumor with thymic epithelial hyperplasia.

Acute alloxan toxicity causes granulomatous tubulointerstitial nephritis with severe mineralization.

Alloxan had been recognized as having a direct nephrotoxic effect different from its diabetogenic action. We encountered previously unreported granulomatous tubulointerstitial nephritis with severe luminal and interstitial mineralization in one diabetic rat after one week of alloxan administration. Histopathologically, many dilated and occluded proximal and distal tubules were segmentally observed in the cortex and outer medulla. The tubular lumen contained minerals and cell debris. Tubular epithelial cells were degenerated and piled up, and they protruded into the lumen, where they enveloped minerals. Mineralization was observed mainly in the tubular lumen, and to some extent in the subepithelium and interstitium. The mineralization beneath the tubular epithelium was often continuous from the subepithelium to the interstitium. In these lesions, the tubular basement membrane was disrupted by mineralization, and a granuloma with multinuclear foreign-body giant cells was formed in the interstitial areas.

Lack of Correlation between Aberrant p16, RAR-ß2, TIMP3, ERCC1, and BRCA1 Protein Expression and Promoter Methylation in Squamous Cell Carcinoma Accompanying Candida albicans-Induced Inflammation.

Hyperplastic candidiasis is characterized by thickening of the mucosal epithelia with Candida albicans infection with occasional progression to squamous cell carcinoma (SCC). C. albicans is a critical factor in tumor development; however, the oncogenic mechanism is unclear. We have previously produced an animal model for hyperplastic candidiasis in the rat forestomach. In the present study, we investigate whether impaired DNA methylation and associated protein expression of tumor suppressor and DNA repair genes are involved in the SCC carcinogenesis process using this hyperplastic candidiasis model. Promoter methylation and protein expression were analyzed by methylation specific PCR and immunohistochemical staining, respectively, of 5 areas in the forestomachs of alloxan-induced diabetic rats with hyperplastic candidiasis: normal squamous epithelia, squamous hyperplasia, squamous hyperplasia adjacent to SCC, squamous hyperplasia transitioning to SCC, and SCC. We observed nuclear p16 overexpression despite increases in p16 gene promoter methylation during the carcinogenic process. TIMP3 and RAR-ß2 promoter methylation progressed until the precancerous stage but disappeared upon malignant transformation. In comparison, TIMP3 protein expression was suppressed during carcinogenesis and RAR-ß2 expression was attenuated in the cytoplasm but enhanced in nuclei. ERCC1 and BRCA1 promoters were not methylated at any stage; however, their protein expression disappeared beginning at hyperplasia and nuclear protein re-expression in SCC was observed only for ERCC1. These results suggest that aberrant p16, RAR-ß2, TIMP3, ERCC1, and BRCA1 expression might occur that is inconsistent with the respective gene promoter methylation status, and that this overexpression might serve to promote the inflammatory carcinogenesis caused by C. albicans infection.

Probiotic (yogurt) containing Lactobacillus gasseri OLL2716 is effective for preventing Candida albicans-induced mucosal inflammation and proliferation in the forestomach of diabetic rats.

Oral and esophageal candidiasis sometimes leads to mucosal hyperplasia, and progresses to carcinoma. We have produced an animal model for hyperplastic mucosal candidiasis in the forestomach that has a proliferative lesion of the squamous epithelium with chronic inflammation and C. albicans infection, some of which advanced to squamous cell carcinoma. There are many reports of the antibacterial effects of probiotics, but consensus about their antifungal effect has not been reached. In the present study, we investigate whether probiotic (yogurt) containing Lactobacillus gasseri OLL2716 (LG21 yogurt) can prevent proliferative and inflammatory changes caused by C. albicans in this mucosal candidiasis animal model. Diabetes was induced in 8-week-old WBN/Kob rats by intravenous administration of alloxan. One group of diabetic rats received a saline containing C. albicans and LG21 yogurt orally (DC+LG21 group) for 30 weeks, and another group received only C. albicans (DC group) for 30 weeks. They were sacrificed at 40 weeks of age, and analyzed histopathologically. In the DC+LG21 group, squamous hyperplasia at the greater curvature was significantly milder, and the Ki-67 positive index was significantly lower compared with the DC group. Suppurative inflammation with C. albicans also tended to be suppressed at the greater curvature. These findings suggest that probiotic (yogurt) containing Lactobacillus gasseri OLL2716 can suppress squamous hyperplastic change and inflammation associated with C. albicans infection in the forestomach.

A novel diabetic murine model of Candida albicans-induced mucosal inflammation and proliferation.

Chronic hyperplastic candidiasis (CHC) lesions will progress to dysplasia with some of these developing squamous cell carcinoma (SCC). It is well known that diabetic patients are predisposed to candidiasis. Previously, we found that alloxan-induced diabetic rats spontaneously have mucosal hyperplasia with C. albicans infection and that those lesions progress to SCC. Here, we developed a rat model of candidiasis with diabetes progressing to mucosal proliferation. Diabetes was induced in thirty rats by single intravenous administration of alloxan. Ten nondiabetic rats and fifteen diabetic rats then received C. albicans containing solution orally, and additional fifteen diabetic rats received saline in the same manner. The administration of C. albicans induced mucosal candidiasis and the related mucosal hyperplastic changes in all the diabetic rats and progressed to SCC in one rat. Chronic suppurative inflammation of the mucosa developed in the forestomach with infection by C. albicans. The same lesions were only detected in the forestomach of 4 diabetic rats without C. albicans treatment. After C. albicans treatment, none of the nondiabetic rats showed mucosal changes or fungus infection in the forestomach. These findings demonstrate that a prolonged diabetic condition can cause C. albicans infection and enhance C. albicans-related mucosal hyperplasia.