Animal PET scanner with a large field of view is suitable for high-throughput scanning of rodents.

OBJECTIVE: In preclinical studies, high-throughput positron emission tomography (PET) imaging, known as simultaneous multiple animal scanning, can reduce the time spent on animal experiments, the cost of PET tracers, and the risk of synthesis of PET tracers. It is well known that the image quality acquired by high-throughput imaging depends on the PET system. Herein, we investigated the influence of large field of view (FOV) PET scanner on high-throughput imaging. METHODS: We investigated the influence of scanning four objects using a small animal PET scanner with a large FOV. We compared the image quality acquired by four objects scanned with the one acquired by one object scanned using phantoms and animals. We assessed the image quality with uniformity, recovery coefficient (RC), and spillover ratio (SOR), which are indicators of image noise, spatial resolution, and quantitative precision, respectively. For the phantom study, we used the NEMA NU 4-2008 image quality phantom and evaluated uniformity, RC, and SOR, and for the animal study, we used Wistar rats and evaluated the spillover in the heart and kidney. RESULTS: In the phantom study, four phantoms had little effect on imaging quality, especially SOR compared with that for one phantom. In the animal study as well, four rats had little effect on spillover from the heart muscle and kidney cortex compared with that for one rat. CONCLUSIONS: This study demonstrated that an animal PET scanner with a large FOV was suitable for high-throughput imaging. Thus, the large FOV PET scanner can support drug discovery and bridging research through rapid pharmacological and pathological evaluation.

Effects of microsampling on toxicity evaluation of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance, in a mouse toxicity study.

ICH S3A Q&A focused on microsampling (MS) was published to help accelerate the use of MS and states that MS is useful because toxicokinetic (TK) evaluation with conventional blood sampling volume requires many animals for TK satellite groups; however, there are few reports of MS application in mice. We investigated the influence of MS on toxicity evaluation in mice by comparing the toxicity parameters with and without MS after a single oral administration of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance. Blood samples (50 µL/point) were collected from the tail vein of 3 mice per group at 2 or 3 time points during a 24-hr period, and toxicity was evaluated 2 days after administration. ANIT-related changes suggesting liver or gallbladder injury were noted in blood chemistry and histopathology. Some of these changes such as increases in focal hepatocyte necrosis and inflammatory cell infiltration in the liver as well as mucosal epithelium necrosis in the gallbladder were apparently influenced by MS. A tendency to anemia was noted in animals with MS but not without MS, which was also noted in the vehicle-treated controls, suggesting influence of blood loss. The current results indicate that ANIT hepatotoxicity could be evaluated in mice in which blood samples were collected by MS for most parameters; however, parameters in anemia and pathology in the liver and gallbladder were influenced by MS in this study condition with ANIT. Therefore, MS application in mice should be carefully considered.

Successful lactation in Plgrkt-deficient female mice caused by a 1-bp deletion of exon4.

Plasminogen (Pg) activation on the cell surface is important for various (patho)physiologic conditions, and Plg-RKT is a cell membrane protein that binds to Pg and promotes its activation. To evaluate the role of Plg-RKT in atherosclerosis, Plgrkt gene in Ldlr-/-/Apobec1-/- was modified using in vivo CRISPR/Cas9. Synthetic RNA for Plgrkt and Cas9 complex was electroporated into the fertilized eggs in the oviducts. Plgrkt deficient mice were established through a 1-bp deletion, and in this research communication we report their lactational ability. In contrast to Plgrkt-/- mice developed by a conventional method, these newly developed mice did not suffer lactation failure and could maintain their pups until weaning. The major obvious difference between these lines is the area of gene modification. The conventionally developed mouse possesses about 10 kb deletion of Plgrkt, which might relate to the lactation failure. Lactation failure is a lethal phenotype in mammals, and analyses of causative genes are especially important for dairy industries. Further genome-wide analyses with both Plgrkt-/- mice may help to establish causative genes for lactation failure.

Inhibition of plasminogen suppresses fibrosis and macrophage foaming in a nonalcoholic steatohepatitis mouse model.

Nonalcoholic steatohepatitis is a clinically important liver disease. Its symptoms are exacerbated by macrophage foaming, which is promoted by plasminogen in vitro. However, the influence of plasminogen on nonalcoholic steatohepatitis has not been reported. In this study, we evaluated the influence of plasminogen in a mouse model of nonalcoholic steatohepatitis with macrophage foaming. L-/- /A-/- mice, characterized by hypercholesterolemia, were injected with streptozotocin and fed a high-fat diet to develop nonalcoholic steatohepatitis with macrophage foaming. To confirm the influence of plasminogen, we used the well-known plasminogen inhibitor tranexamic acid and L-/- /A-/- /Plg-/- mice, which are deficient in plasminogen and investigated the influence on nonalcoholic steatohepatitis. The influence of plasminogen on the expression levels of proinflammatory cytokines involved in foaming in macrophages was also assessed. The formation of nonalcoholic steatohepatitis lesions with macrophage foaming was confirmed in the L-/- /A-/- mouse model. Tranexamic acid attenuated foaming and fibrosis in the L-/- /A-/- mice. Similarly, foaming and liver fibrosis were also attenuated in the L-/- /A-/- /Plg-/- mice. The mRNA expression levels of TGF-ß1 and IL-1ß in liver and peritoneal macrophages were reduced upon plasminogen inhibition. We show that inhibition of plasminogen suppressed macrophage foaming, cytokine expression, and consequently fibrosis in nonalcoholic steatohepatitis. Our results provide a clue toward various processes leading to fibrosis and may contribute to new therapeutic strategies for nonalcoholic steatohepatitis.

Immunohistochemical Characteristics of Thymomas and Hyperplastic Lesions in Wistar Hannover Rats.

Previously, we investigated the higher incidence of hyperplastic lesions and thymomas and histopathological resemblance of cortex-medullary structures between thymomas and normal thymuses in Wistar Hannover (WH) rats. Thymomas had pale-staining cell foci (PA) similar to medulla but without lymphocytes. Here, we focused on the differences in cytokeratin (CK) expression in the thymic epithelia of the cortex and medulla and compared the structures of thymomas and normal thymuses. Thymomas, hyperplastic lesions, and normal thymuses obtained from background studies of WH rats were stained with antibodies against CK14, CK18, and CD20. In normal thymuses, the epithelial cells were positive for CK14 in the medulla and subcapsular area and for CK18 in the cortex, B-cells were positive for CD20 in the medulla. In thymomas, the epithelial cells were positive for CK14 in the medullary differentiation (MD) areas and for CK18 in the cortex-like lymphocyte rich and PA, and B-cells were positive for CD20 in the MD areas.

Spontaneous globule leukocyte tumor accompanied by inflammatory cells in a Wistar Hannover rat.

We encountered hematolymphoid neoplastic lesions in the form of many nodules in the spleen and liver in a 110-week-old male Wistar Hannover rat (Crl:WI (Han)). The lesions contained atypical proliferative cells, eosinophils, lymphocytes, and macrophages. The proliferative cells comprised various atypical cell types with or without cytoplasmic eosinophilic granules. The granules were positively stained using periodic acid-Schiff and elastase stains, were bluish purple using phosphotungstic acid and hematoxylin, and showed no metachromasia using toluidine blue. In immunohistochemical staining, the proliferative cells with or without granules were positive for granzyme B, rat mast cell protease II, and Ki67. Electron microscopic examination revealed that single to multiple high-density granules of variable size were covered by a membrane. These findings led to a diagnosis of globule leukocyte tumor. The accompaniment of this tumor by inflammatory cells is likely evoked by mast cell-like active mediators contained in the granules of the globule leukocytes.

Supplemental study on 2', 3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNPase) activity in developing rat spinal cord lesions induced by hexachlorophene and cuprizone.

In a previous study, we showed that 2', 3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNPase) expression is induced in different temporal patterns in the cerebrum, cerebellum and medulla oblongata of hexachlorophene (HCP) and cuprizone (CPZ) treated rats. Here, we additionally examined the histopathological changes and CNPase expression in the spinal cord to clarify the reproducibility of different temporal patterns of CNPase expression in the spinal cord showing low degree or lack of spongy changes. Spongy changes were observed in HCP-treated rats, but not in CPZ-treated rats. Immunohistochemistry showed that intense expression of CNPase was not induced following HCP or CPZ treatment. Our data reveal that expression intensity of CNPase may be dependent on the degree of HCP- and CPZ-induced damage of the myelin sheath.

Thymomas and Associated Hyperplastic Lesions in Wistar Hannover Rats.

Thymomas occur prevalently in aged Wistar Hannover (WH) rats, along with hyperplastic lesions that cannot be categorized as thymomas. We compared the histological features of hyperplastic lesions and thymomas in WH rats, the incidences of these lesions, and the relationship of these lesions to the degree of thymic involution and also compared these lesions with those of Sprague Dawley (SD) rats in 4-, 13-, 26-, and 104-week studies. There were no morphological differences between hyperplastic cells and benign tumor cells in thymomas. The histological difference between hyperplastic lesions and thymomas was the size of the proliferative areas and the number of medullary differentiation areas. The hyperplastic lesions of the thymus in WH rats might have a potential for progression to thymomas due to the observed multiple hyperplastic lesions or mixed lesions with thymomas. The incidence of these proliferative lesions in the thymus was higher in females than in males. Further, the incidence of these proliferative lesions was higher in WH rats than in SD rats. Thymic involution was more severe in males than in females and more severe in SD rats than in WH rats. The differences in involution progression may have been reflected in the incidence of thymic proliferative lesions in SD and WH rats.

Morphological and immunohistochemical diversity of endometrial stromal sarcoma in rats.

To clarify the histopathological characteristics of rat endometrial stromal sarcoma (ESS), we morphologically reviewed 12 malignant uterine tumors protruding into the lumen in previous rat carcinogenicity studies. The 12 cases were classified into the following 6 types based on their morphological features: spindle cell and collagen rich type, pleomorphic/spindle cell and compact type, decidual alteration type, histiocytic and multinucleated giant cell mixture type, Antoni A-type schwannoma type, and Antoni B-type schwannoma type. Immunohistochemically, tumor cells in all cases exhibited focal or diffuse positive reactions for vimentin, and 11 of the 12 cases were positive for S-100. Interestingly, 9 cases were positive for desmin or αSMA, indicating tumor cells expressing smooth muscle properties. Both Antoni A- and B-type schwannoma types showed low reactions for both muscle markers. Positive results for estrogen receptor α in the 11 cases suggested that they were derived from endometrial stromal cells. On the basis of their immunohistochemical profiles, they were considered to be derived from endometrial stromal cells while they showed morphological variation. The detection of a basement membrane surrounding tumor cells might not be a definitive indicator for differential diagnosis of ESS from malignant schwannoma. In conclusion, ESS could exhibit wide morphological and immunohistochemical variation including features of schwannoma or smooth muscle tumor.

A case report of ventricular septal defect in an adult Sprague Dawley rat.

A 20-week-old male Sprague Dawley rat noted with decreased body weight, dyspnea, and anorexia beginning 2 days before death was necropsied in the recovery period of a sub-chronic toxicity study. The heart was severely enlarged (30 × 20 × 20 mm), 3-4 times larger than normal, with an approximately 6 mm wide defect in the upper, membranous portion of the ventricular septum. Both ventricles measured 4 mm in thickness, and the right ventricle was 4 times thicker than normal. According to a microscopic examination, the myocardial fibers were severely hypertrophic in the right ventricle and mildly hypertrophic in the left ventricle and septum. Myocardial vacuolation, focal hemorrhages with hemosiderin-laden macrophages, myocardial necrosis, focal fibrosis, hyalinized myocardial fibers, and multifocal adhesive pericarditis were also present. This is the first report concerning severe ventricular septal defects in an adult Sprague Dawley rat with a detailed histopathological examination.

Dosimetry for lung tumorigenesis induced by urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P) in A/JJmsSlc mice.

Some chemicals are known to be lung carcinogens in rodents. While many studies using two-stage models have administered medium or high doses to mice, few have tested lower doses. The dose dependence of urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P), three well-known lung carcinogens at high doses, has not been sufficiently reported in lower dose ranges. Our study evaluated the tumorigenicity of urethane, NNK, and B[a]P at 26 weeks after a single intraperitoneal administration of each compound within medium to low dose in male and/or female A/JJmsSlc (A/J) mice. Dose-dependent tumorigenesis was demonstrated histopathologically for the three compounds. These results suggested that the tumorigenicity of these chemicals is dose dependent in A/J mice, even at lower doses than previously reported.

Lymphocytic adrenal medullitis and lymphocytic thyroiditis in a laboratory beagle dog.

Lymphocytic adrenal medullitis characterized by inflammation and atrophy in the medulla of the bilateral adrenal glands was observed in an 18-month-old male laboratory beagle dog. It might be that the present lymphocytic adrenal medullitis is an autoimmune-mediated disease as the histological characteristics are consistent with an autoimmune pathogenesis. However, the actual cause remains unclear as the existence of serum autoantibodies against the adrenal medulla could not be confirmed. Although this dog also contracted lymphocytic thyroiditis along with serum thyroglobulin autoantibodies, indicating that the thyroiditis occurred with an autoimmune basis; the relation between the adrenal medullitis and thyroiditis is unknown.

Spontaneous Age-related Lesions of the Kidney Fornices in Sprague-Dawley Rats.

The upper portion of the rat kidney pelvis has specialized anatomic structures referred to as fornices. Fornices have a role in urine concentration. Spontaneous lesions including mineralization, epithelial hyperplasia, and inflammatory cell infiltration may occur in the area of the fornices. However, little information regarding specific historical control data or the spontaneous development of these findings in male and female fornices is known. Understanding spontaneous age-related lesions in the area of the fornices versus other portions of the kidney pelvis may be relevant in the identification of test article-induced changes. A retrospective study was conducted of male and female Sprague-Dawley rat kidney fornices over several time points to determine the incidence and severity of mineralization, epithelial hyperplasia, and inflammatory cell infiltration. Based on this investigation, these lesions appeared to increase over time and, in general, occurred earlier and with a greater incidence in females. Regarding those chemicals that may result in lesions of the kidney pelvis, it may be important for pathologists to separately diagnose lesions of the fornices from other portions of the kidney pelvis to help differentiate between any spontaneous age-related and induced changes.

An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat.

Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors.

Bacterial pleuritis with thickened mesothelial hyperplasia in a young beagle dog.

A five-month-old male beagle dog suddenly became moribund. Bloody fluid accumulated in the thoracic and abdominal cavities, and soft yellow flecks were floating in the thoracic fluid. The mediastinum and pericardium became dark reddish with villous thickening. Other parietal and pulmonary pleurae were rough, and the organs adhered to each other. Histologically, most mediastinal pleura formed papillary projections covered by a single layer of mesothelial cells. Many macrophages and neutrophils infiltrated the submesothelial connective tissue. At the mediastinum adjacent to the pericardium, cuboidal mesothelial cells proliferated solidly and formed a thick surface stratum. The flecks consisted of gram-negative filamentous or small bacillary (coccoid) bacteria. In the right posterior lobe of the lung, neutrophilic infiltration and a large encapsulated abscess including a bacterial colony were present. We diagnosed this case as "bacterial pleuritis with thickened mesothelial hyperplasia". The cause of the pleuritis might be a chronic pleural infection spread via the lung abscess.

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.

Malignant Mesothelioma in the Thoracic Cavity of a Crj:CD(SD) Rat Characterized by Round Hyalinous Stroma.

Spontaneous malignant mesothelioma was found in a 104-week-old male Crj:CD(SD) rat. The tumor was scattered on the surface of the lung, heart, mediastinal pleura and thoracic wall and metastasized to the alveolar septa. Histopathologically, small flattened or cuboidal tumor cells proliferated with stroma, formed almost normal papillary structures and reacted positively to colloidal iron stain and immunohistochemical staining for mesothelin. Round hyalinous stromata were pronounced, which is a characteristic feature, and the possible reason for this is as follows; at first, a small amount of collagen fibers was formed in the center of the clusters of several tumor cells, and then the cell clusters expanded like balloons with an increase in the collagen fibers.

Effect of di(2-ethylhexyl) phthalate (DEHP) on genital organs from juvenile common marmosets: I. Morphological and biochemical investigation in 65-week toxicity study.

Recent studies demonstrated that preadolescent male rats are more sensitive to testicular damage from exposure to DEHP than adults. Male and female marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo). No treatment-related changes were observed in male organ weights, and no microscopic changes were found in male gonads or secondary sex organs. Sperm head counts, zinc levels, glutathione levels, and testicular enzyme activities were comparable between groups. Electron microscopic examination revealed no treatment-related abnormalities in Leydig, Sertoli, or spermatogenic cells. Histochemical examination of the testis after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alterations in steroid synthesis in the Leydig cells. Thus, although marmoset monkeys were treated with 2500 mg/kg DEHP, throughout the pre- and periadolescent period, no histological changes were noted in the testes. For females, increased ovarian and uterine weights and elevated blood estradiol level were observed in higher dosage groups, 500 and 2500 mg/kg. These increased weights were associated with the presence of large corpus luteum, a common finding in older female marmosets. Although an effect on the female ovary cannot be completely ruled out, no abnormal histological changes were observed in the ovaries or uteri in comparison to controls. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6beta-hydroxylase, and lauric acid omega-1omega-hydroxylase activities) were increased in males and/or females of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae.

The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.