Naho Tsuji

Organogenesis of mild ocular coloboma in FLS mice: Failure of basement membrane disintegration at optic fissure margins

Fatty Liver Shionogi (FLS) mice have been shown to develop a hereditary disorder characterized by localized retinochoroidal defects of the ventral fundus very similar to human typical ocular coloboma without microphthalmia. The objective of this study was to determine when and how the failure of the optic fissure closure occurs, and to clarify the disturbed mechanism of basement membrane disintegration during embryonal stage in FLS mice. Fetuses at day 11.5-15.5 of gestation were obtained from dams of FLS and BALB/c strain of mice. Coronal serial sections through the eye were examined by light and electron microscopy. The sections were followed by observation of the basement membrane using reaction with periodic acid-Schiff (PAS) reagent and immunohistochemical staining with anti-Laminin and anti-Type IV collagen antibodies. Both optic fissure margins closely approached each other up to GD 11.5 in all FLS and BALB/c embryos. The inner and outer layers of the optic cup did not normally fuse at midlenticular levels of the optic fissure in almost 70% of FLS fetuses by GD 15.5, whereas both margins were completely fused in all BALB/c fetuses of the same gestational day. In the FLS fetuses at GD 12.5, rolling on one side of fissure margins and consequent asymmetry were observed at the ventral optic fissure. The basement membrane persisted after the close contact of both sides of the fissure margins during GD 11.5 and 15.5. Ultrastructurally, the basal lamina was not disintegrated and mesenchymal cells intervened between the two neuroepithelial layers, resulting in complete separation of both fissure margins at GD 13.0. It is highly probable that the disturbed basement membrane disintegration right before optic fissure closure causes mild ocular coloboma without microphthalmia in FLS mice.

Carnitine is necessary to maintain the phenotype and function of brown adipose tissue

The juvenile visceral steatosis (JVS) mouse is a mutant strain with an inherited systemic carnitine deficiency. Mice of this strain show clinical signs attributable to impaired heat production and disturbed energy production. Brown adipose tissue (BAT) is the primary site of non-shivering thermogenesis in the presence of uncoupling protein-1 (UCP-1) in rodents and humans, especially in infants. To investigate the possible cause of impaired heat production in BAT, we studied the morphological features, carnitine concentration, and UCP-1 production of BAT in JVS mice. The effect of carnitine administration on these parameters was also examined. JVS mice aged 5 or 10 days (60 each) and age-matched control mice were used in this study, along with 10-day-old JVS mice treated subcutaneously with L-carnitine once a day between postpartum days 5 and 10. JVS mice showed lower body temperatures and lower concentrations of carnitine in BAT. Morphologically, BAT cells in JVS mice contained large lipid vacuoles and small mitochondria, similar to those present in white adipose tissue cells. In addition, UCP-1 mRNA and protein expression levels were significantly reduced in JVS as compared with control mice. Carnitine treatment resulted in significant increases in body temperature and carnitine concentrations in BAT, together with the recovery of normal morphological features. UCP-1 mRNA and protein expression levels were also significantly increased. These findings strongly suggest that carnitine is essential for maintaining the function and morphology of BAT.

Cutaneous Hemangiosarcoma in a Dog

A male golden retriever of unknown age presented with multiple cutaneous and subcutaneous masses from the left elbow to the digits. Histopathologically, multiple tumor foci had formed from the dermis to the subcutaneous tissue. Tumor foci consisted of a vascular structure, alveolar structure and solid proliferative area. The borders among these areas were not clear. Some neoplastic cells resembled a mature endothelium, while others were large pleomorphic cells. Immunohistochemically, the tumor cells were usually strongly positive for CD31 and often positive for PROX-1, the lymphatic endothelial cell marker. Based on these findings, the tumor was diagnosed as a hemangiosarcoma with lymphatic differentiation.

Macrophage-Associated Gelatinase Degrades Basement Membrane at the Optic Fissure Margins During Normal Ocular Development in Mice

Purpose Basement membrane degradation and macrophage aggregation at the optic fissure margins are crucial to optic fissure closure during normal murine eye development. Basement membrane degradation is also an essential step in cancer development, and matrix metalloproteinases (MMPs) play an important role. In this study, we investigated MMP alteration at the degrading basement membrane of optic fissure margins in mice and attempted to clarify the relationship between MMP activity and macrophages. Methods Serial coronal frozen sections of eyes from BALB/c fetuses were prepared and gelatinase activity was examined using in situ zymography techniques. The frozen sections were immunohistochemically stained with anti-F4/80, anti-MMP 2, and anti-MMP 9 antibodies. Serial coronal paraffin sections were also immunohistochemically stained with anti-type IV collagen and anti-F4/80, and basement membrane disintegration and macrophage aggregation at the optic fissure margins were examined. Results The basement membrane of optic fissure margins was rapidly degraded during gestational days (GDs) 12.0 to 12.5. Meanwhile, gelatinase activity at F4/80-positive macrophages significantly increased during GDs 11.5 to 12.0 and declined thereafter; some of those were also positive for MMP2. The number of macrophages was also increased and decreased at nearly the same time. Conclusions Intramacrophage MMPs may be responsible for basement membrane degradation at the optic fissure margins during normal eye development in mice.

Histopathology of a wavy medaka

Wavy medakas are medakas that exhibit spinal curvature characterized by dorsoventrally curved vertebrae. We found a spontaneous wavy medaka in our experimental stock and subjected it to a histopathological examination. Macroscopically, the wavy medaka’s spine formed an M shape, and its vertebrae displayed a dorsoventral curvature that started at the third vertebral bone. Microscopically, the vertebral cavities were filled with fibrous tissue, which was similar to that seen in the central parts of the intervertebral discs of a normal medaka. The vertebral joints were composed of vacuolated notochord cells without intervertebral disc formation. These changes were also observed in the caudal region, which exhibited less curvature. In the normal medaka, the intervertebral discs form via the regression of the notochord that plays a key role in the development of vertebrae and disc formation. We concluded that notochordal subinvolution had induced intervertebral disc dysplasia, leading to lordokyphosis, in the wavy medaka.

Histomorphological comparison of rat placentas by different timing of chlorpromazine-administration.

The effects of chlorpromazine-treatment timing on the development of the placenta in pregnant rats were examined. Chlorpromazine was administered intraperitoneally at 100mg/kg on gestation day (GD) 11 (GD11-treated group), GD 13 (GD13-treated group) or GD 15 (GD15-treated group) into pregnant rats. All treated dams exhibited decreased body weight, prone position, hypothermia, loss or decrease of locomotor activity, etc. The fetal mortality rates were increased up to 42.9% in the GD11- and GD13-treated groups and up to 16.7% in the GD15-treated group. The embryo/fetal weight was on a declining trend from GD 16 onward, and the intrauterine growth retardation (IUGR) rates on GD 21 were increased in all treated groups. The placental weight showed a declining trend from GD 15 onward in all treated groups. Histopathologically, apoptosis was detected 1 or 2 days after treatment, and led to hypoplasia in the labyrinth zone and metrial gland, and cystic degeneration in the basal zone on GD 21 in all treated groups. There was no difference in the histopathological lesions on GD 21 among the treated groups. Thus, it is considered that chlorpromazine-induced placental toxicity is characterized in that there is no obvious specific sensitive period from GD 11 to GD 15. Chlorpromazine induced a non-specific transient development retardation of the placenta by apoptosis independently of the cell proliferation period in each part/zone.

Morphology and physiology of rat placenta for toxicological evaluation

The placenta plays a pivotal role in fetal growth, and placental dysfunction and injury are associated with embryo/fetal toxicity. Histological examination of the rat placenta for safety evaluation provides valuable clues to the mechanisms of this toxicity. However, the placenta has specific and complex biological features unlike those of other organs, and placental structure dramatically changes depending on the time during the gestation period. Thus, time-dependent histopathological examination of the rat placenta should be performed based on the understanding of normal developmental changes in morphology and function. The placentas of rats and humans are both anatomically classified as discoid and hemochorial types. However, there are differences between rats and humans in terms of placental histological structure, the fetal-maternal interface, and the function of the yolk sac. Therefore, extrapolation of placental toxicity from rats to humans should be done cautiously in the evaluation of risk factors. This review describes the development, morphology, physiology, and toxicological features of the rat placenta and the differences between the rat and human placenta to enable accurate evaluation of reproductive and developmental toxicity in studies.

Effect of dibutyltin on placental and fetal toxicity in rat

In order to elucidate the effect of chorioallantoic and yolk sac placenta on the embryonic/fetal toxicity in dibutyltin dichloride (DBTCl)-exposed rats, we examined the histopathological changes and the tissue distribution of dibutyltin in the placentas and embryos. DBTCl was orally administered to the groups at doses of 0 mg/kg during gestation days (GD)s 7-9 (control group) and 20 mg/kg during GDs 7-9 (GD7-9 treated group), and GDs 10-12 (GD10-12 treated group). The total fetal mortality was increased, and malformations characterized by craniofacial dysmorphism were detected in the GD7-9 treated group. The embryonic/fetal weight and placental weight showed a decrease in both DBTCl-treated groups. Histologically, some embryos on GD 9.5 in the GD7-9 treated group underwent apoptosis without any changes of yolk sac. In the laser ablation-inductively coupled plasma-mass spectrometry analysis (LA-ICP-MS), tin was detected in the embryo, allantois, yolk sac, ectoplacental cone and decidual mass surrounding the conceptus on GD 9.5 in the GD7-9 treated group. Thus, it is considered that the embryo in this period is specifically sensitive to DBTCl-induced apoptosis, compared with other parts. The chorioallantoic placentas in both DBTCl-treated groups showed the developmental delay and hypoplasia in the fetal parts of placenta, resulting from apoptosis and mitotic inhibition. Thus, it was speculated that the DBTCl-induced malformations and fetal resorption resulted from the apoptosis in the embryo caused by the direct effect of DBTCl. The DBTCl-induced lesions in the chorioallantoic placenta were a non-specific transient developmental retardation in the fetal parts of placenta, leading to intrauterine growth retardation.

Iridal coloboma induces dyscoria during miosis in FLS mice.

OBJECTIVE Fatty liver Shionogi (FLS) mice exhibit characteristic retinochoroidal coloboma because of a failure in fusion of the embryonic optic fissure. However, the same pathogenesis should result in iridal coloboma that has not been reported in this strain. The purpose of this study was to describe the physiologic and morphometric changes in iridal tissue involved in ocular coloboma in FLS mice. PROCEDURES The miotic response after light exposure was evaluated in three strains of live mice, and the shape and location of the pupil were judged macroscopically. Subsequently, macroscopic abnormalities in the anterior segment and fundus were observed postmortem in all mice. During miotic and mydriatic responses in the eyes of live male FLS mice with dyscoric and normal pupils, each iris was measured in four radial directions. The enucleated eyes were examined morphometrically and histologically in both sexes of FLS mice. RESULTS Inferior corectopia upon light-induced miosis was clearly detected in live FLS mice. The deviated pupils were not round but oval-shaped. Clinical and postmortem examination revealed that all dyscoric eyes had hypoplastic and dysfunctional irides inferiorly in FLS mice. Histopathological examination confirmed that both the dilator and sphincter muscles and iris stroma were quantitatively diminished in the affected inferior iris. Meanwhile, the rate of fundus (retinochoroidal) coloboma in eyes exhibiting dyscoria was remarkably high, although some dyscoric eyes had no fundus coloboma. CONCLUSIONS Fatty liver Shionogi mice had iridal coloboma, resulting in inferior corectopia upon light-induced miosis as an indicator of ocular coloboma.