Effect of deoxycorticosterone acetate-salt-induced hypertension on diabetic peripheral neuropathy in alloxan-induced diabetic WBN/Kob rats

The relationship between hypertension and diabetic peripheral neuropathy (DPN) has recently been reported in clinical research, but it remains unclear whether hypertension is a risk factor for DPN. To investigate the effects of hypertension on DPN, we analyzed morphological features of peripheral nerves in diabetic rats with hypertension. Male WBN/Kob rats were divided into 2 groups: alloxan-induced diabetic rats with deoxycorticosterone acetate-salt (DOCA-salt) treatment (ADN group) and nondiabetic rats with DOCA-salt treatment (DN group). Sciatic, tibial (motor) and sural (sensory) nerves were subjected to qualitative and quantitative histomorphological analysis. Systolic blood pressure in the two groups exhibited a higher value (>140 mmHg), but there was no significant difference between the two groups. Endoneurial blood vessels in both groups presented endothelial hypertrophy and narrowing of the vascular lumen. Electron microscopically, duplication of basal lamina surrounding the endothelium and pericyte of the endoneurial vessels was observed, and this lesion appeared to be more frequent and severe in the ADN group than the DN group. Many nerve fibers of the ADN and DN groups showed an almost normal appearance, whereas morphometrical analysis of the tibial nerve showed a significant shift to smaller fiber and myelin sizes in the ADN group compared with DN group. In sural nerve, the fiber and axon-size significantly shifted to a smaller size in ADN group compared with the DN group. These results suggest that combined diabetes and hypertension could induce mild peripheral nerve lesions with vascular changes.     

Malignant Lymphoma with Severe Infiltrative Growth into Skeletal Muscles in WBN/Kob Rats

Although spontaneously occurring neoplasms have been reported repeatedly in F344, SD and Wistar rats, which are commonly used strains for routine toxicologic and carcinogenicity studies, there are only a few reports of malignant lymphoma or lymphatic leukemia except for large granular lymphocytic leukemia (LGL) in F344 rats. Malignant lymphoma (lymphosarcoma) is thought to be uncommon in F344 rats. The authors encountered malignant lymphomas of the non-LGL leukemia type with characteristic pathologic features in WBN/Kob rats. The mean age at onset of the disease in all 13 affected rats (8 males and 5 females) was about 60 weeks. Common and characteristic clinical signs were abnormal gait with hind limb paralysis. Macroscopically, the enlargement of the lymph nodes, spleen and liver was slight to moderate. Scattered multiple white-to-gray nodules encompassed the aorta and assumed a bead-like appearance near the thoracic and lumbar vertebrae. Histopathologically, neoplastic proliferative changes were predominant in the bone marrow tissue of the entire body, and many tumor cells infiltrated the spleen and several lymph nodes. The most striking histological features were constant and severe infiltration of tumor cells in the adipose tissue and skeletal muscle adjacent the thoracic and lumber vertebrae. Immunohistochemically, all tumor cells were positive for B-cell markers (PAX-5, CD79a and CD45) and negative for CD3. From the results of immunohistochemistry and morphological examination, these tumors were diagnosed as malignant B-cell lymphomas.     

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.     

Bone morphological analyses in Spontaneously Diabetic Torii (SDT) fatty rats

The Spontaneously Diabetic Torii (SDT) fatty rat, a model for obese type 2 diabetes, shows bone quantitative abnormalities, namely low bone mineral density (BMD). The objective of this study was to evaluate bone morphological changes, in particular identifying the bone qualitative abnormalities, in the SDT fatty rat. Male SDT fatty rats showed increases in total trabecular area and trabecular number and decreases in trabecular thickness in cancellous bones of the proximal tibia, indicating trabecular miniaturization. The SDT fatty rat is useful for investigation of pathophysiological changes in bone quality in diabetic osteoporosis.     

Histopathological nerve and skeletal muscle changes in rats subjected to persistent insulin-induced hypoglycemia

New insulin analogues with a longer duration of action and a flatter pharmacodynamic profile are developed to improve convenience and safety for diabetic patients. During the nonclinical development of such analogues, safety studies must be conducted in nondiabetic rats, which consequently are rendered chronically hypoglycemic. A rat comparator model using human insulin would be valuable, as it would enable differentiation between effects related to either persistent insulin-induced hypoglycemia (IIH) or a new analogue per se. Such a model could alleviate the need for an in-study-comparator and thereby reduce the number of animals used during development. Thus, the aims of the present study were i) to develop a preclinical animal model of persistent hypoglycemia in rats using human insulin infusion for four weeks and ii) to investigate histopathological changes in sciatic nerves and quadriceps femoris muscle tissue, as little is known about the response to persistent hypoglycemia in these tissues. Histopathologic changes in insulin-infused animals included axonal degeneration and myofibre degeneration. To our knowledge, this is the first study to show that persistent IIH provokes peripheral nerve and skeletal myofiber degeneration within the same animals. This suggests that the model can serve as a nonclinical comparator model during development of long-acting insulin analogues.     

Effects of preventing hyperphagia on glycolipid metabolic abnormalities in Spontaneously Diabetic Torii fatty rats

Spontaneously Diabetic Torii (SDT) fatty rats, made by introducing the fa allele of the Zucker fatty rat into the SDT rat genome, represent a new model of obese type 2 diabetes. SDT fatty ^fa/fa (SDT fatty) rats exhibit overt obesity, hyperglycemia, and hyperlipidemia from about six weeks of age, and this is associated with hyperphagia by an induced disorder of leptin action. The present study was conducted to elucidate whether suppression of hyperphagia can improve reduce abnormalities in SDT fatty rats. SDT fatty rats were subjected to pair-feeding with SDT fatty ^{+/ +} (SDT) rats from 6 to 26 weeks of age, and the effects on metabolic parameters and diabetic complications were assessed. Body weights of the pair-fed rats were similar with those of SDT rats during the experimental period. Improvement of hyperglycemia or hypertriglyceridemia was observed from 8 to 16 or 12 weeks of age in the pair-fed rats, but hypercholesterolemia was not entirely improved during the experimental period. We also examined mRNAs expression in liver, and found that the expression associated with glyconeogenesis, such as glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), tended to decrease in the pair-fed rats, and the mRNA expression of hydroxymethylglutaryl-CoA (HMG-CoA) was elevated. Renal parameters, such as blood urea nitrogen and urinary albumin excretion, were improved in the pair-fed rats. The incidence or progression of diabetic complications, such as renal lesions and cataract, was reduced. In conclusion, suppression of hyperphagia in SDT fatty rats was effective in temporally improving hyperglycemia or hypertriglyceridemia, and reducing the incidence or progression of diabetic complications, but was ineffective in reducing hypercholesterolemia.     

Histopathological and Immunohistochemical Characterization of Spontaneously Occurring Uterine Deciduomas in Young Adult Rats

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.     

糖尿病大鼠皮肤溃疡阴证模型

糖尿病足溃疡在糖尿病并发症中较为常见且具有高的致死率,引起国内外专家关注。随着研究的深入,中西医在临床中发现一类症状相似糖尿病足溃疡久治难愈,符合中医学的阴证疮面,故称糖尿病足溃疡阴证。要想对糖尿病足溃疡阴证的发病机制,病理特点和干预治疗等多方面有深入的了解,动物模型的建立为其提供可靠有效的途径。论文概括了前人对于糖尿病大鼠皮肤溃疡模型的进展,同时总结其阴证模型的优缺点和自己的经验与认识,以期对临床具有指导意义的糖尿病大鼠皮肤溃疡阴证模型。     

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m³ MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m³ MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m³ MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m³ MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m³.     

Toxicity of Nicotine by Repeated Intratracheal Instillation to F344 Rats

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.     

Transition of Historical Control Data for High Incidence Tumors in F344 Rats

Historical control data of tumor incidence were collected from the control groups (215 animals of each sex) in four recent carcinogenicity studies that were started between 2005 to 2009 (terminally sacrificed between 2007 and 2011) at BoZo Research Center Inc. (Gotemba, Shizuoka, Japan) using Fischer 344 rats (F344/DuCrlCrlj). These data were compared to the previous historical control data (from 1990 to 2004, previously reported) in the same facility. In the results, the incidence of C-cell adenoma in the thyroid tended to increase in both sexes in recent years (30.8% for males and 24.4% for females in 2005-2009) as compared with the previous data (17.4% and 20.1% for males and 11.5% and 11.8% for females in 1990–1999 and 2000–2004, respectively). In addition, the incidences of pancreatic islet cell adenoma in males and uterine adenocarcinoma tended to increase from around 2000 and remained high in recent years (incidences of islet cell adenoma in males of 10.5%, 17.1% and 20.5% in 1990–1999, 2000–2004 and 2005–2009; incidences of uterine adenocarcinoma of 3.3%, 12.0% and 13.5% in 1990–1999, 2000–2004 and 2005–2009, respectively). There was no apparent difference in the incidence of other tumors.     

Occurrence of Spontaneous Tumors in the Central Nervous System (CNS) of F344 and SD Rats

In order to accurately assess the carcinogenicity of chemicals with regard to rare tumors such as rat CNS tumors, sufficient information about spontaneous tumors are very important. This paper presents the data on the type, incidence and detected age of CNS tumors in F344/DuCrlCrlj (a total of 1363 males and 1363 females) and Crl:CD(SD) rats (a total of 1650 males and 1705 females) collected from in-house background data-collection studies and control groups of carcinogenicity studies at our laboratory, together with those previously reported in F344 and SD rats. The present data on F344/DuCrlCrlj rats (F344 rats) and Crl:CD(SD) rats (SD rats) clarified the following. (1) The incidences of all CNS tumors observed in F344 rats were less than 1%. (2) The incidences of malignant astrocytoma and granular cell tumor were higher in male SD rats than in female SD rats. (3) The incidences of astrocytoma and granular cell tumor were higher in SD rats than in F344 rats. (4) Among astrocytoma, oligodendroglioma and granular cell tumor, oligodendroglioma was detected at the youngest age, followed by astrocytoma, and ultimately, granular cell tumor developed in both strains. The incidences observed in our study were almost consistent with those previously reported in F344 and SD rats.     

Chronic Peripheral Administration of Kappa-Opioid Receptor Antagonist Advances Puberty Onset Associated with Acceleration of Pulsatile Luteinizing Hormone Secretion in Female Rats

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.     

Age-related Histological Findings in the Pineal Gland of Crl:CD(SD) Rats

To provide background data as the pathologic basis, the pineal glands of 190 male and 193 female Crl:CD(SD) rats at ages of 0–7, 51–58, 70–85 and 111 weeks were examined histologically in this study. Mineralization and fibrosis were common findings in the aged rats, whereas they were rarely found in the young ones; mineralization was present in 7, 44, 67 and 79% of males and in 0, 32, 67 and 79% in females, and fibrosis was present in 0, 29, 48 and 44% of males and 0, 18, 40 and 35% of females at ages of 0–7, 51–58, 70–85 and 111 weeks, respectively. Striated muscle fiber appeared regularly in the fibrosis region from 51–58 weeks of age when fibrosis increased, while the origin of this fiber remained unclear. Vacuolation of pineal cells also increased with age in both sexes, though the total incidence was low. There was a low incidence of lymphocytic infiltration in both sexes, but this was not related to age.     

Stimulation of Adrenal Chromaffin Cell Proliferation by Hypercalcemia Induced by Intravenous Infusion of Calcium Gluconate in Rats

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.     

Relationship between anti-insulin antibody production and severe insulin resistance in a diabetic cat

A 5-year-old castrated male domestic shorthair cat was diagnosed with diabetic ketoacidosis and severe insulin resistance. Although the conventional treatment for diabetic ketoacidosis was provided, the cat required frequent hospitalization because of severe dehydration and repeated diabetic ketoacidosis. We detected anti-insulin antibodies for human in this cat. Serum insulin-binding IgG levels were markedly elevated compared with those in healthy cats and other diabetic cats. We initiated prednisolone to suppress the effects of anti-insulin antibodies. After initiation of prednisolone, the cat was gradually recovered with increasing activity and appetite. Furthermore, satisfactory glycemic control was achieved with combined subcutaneous injection of insulin detemir and insulin degludec.