Mineo Yasuda

Loss of teratogenic response to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in mice lacking the Ah (dioxin) receptor

The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand‐activated transcription factor that is considered to mediate pleiotropic biological responses such as teratogenesis, tumour promotion, epithelial hyperplasia and the induction of drug‐metabolizing enzymes to environmental contaminants usually represented by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). In contrast to the role of AhR in the regulatory mechanism of xenobiotic‐metabolizing enzymes, there is no direct proof that the AhR is involved in the teratogenic effects of TCDD.

Terminology of developmental abnormalities in common laboratory mammals (version 1)

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

The anatomic relation among the nerve roots, intervertebral foramina, and intervertebral discs of the cervical spine

Study Design. An anatomic study of the cervical intervertebral foramina, nerve roots, and intradural rootlets performed using a surgical microscope. Objectives. To investigate the anatomy of cervical root compression, and to obtain the anatomic findings related to cervical foraminotomy for the treatment of cervical radiculopathy. Summary of Background Data. Cervical foraminotomy is a procedure performed frequently for the management of cervical radiculopathy. However, anatomic studies of cervical foraminotomy have not been fully elucidated. Methods. In this study, 18 cadavers were obtained for the study of the cervical spine. All the soft tissues were dissected from the cervical spine. Thereafter, laminectomy and facetectomy were performed on C4 through T1 using a surgical microscope. The nerve roots and surrounding anatomic structures, including intervertebral discs and foramina, were exposed. In addition, the intradural rootlets and their intersegmental connections were observed. Results. The shape of the intervertebral foramina approximated a funnel, the entrance zone being the most narrow part and the root sleeves conical, with their takeoff points from the central dural sac being the largest part. Therefore, compression of the nerve roots occurred at the entrance zone of the intervertebral foramina. Anteriorly, compression of the nerve roots was caused by protruding discs and osteophytes of the uncovertebral region, whereas the superior articular process, the ligamentum flavum, and the periradicular fibrous tissues affected the nerve posteriorly. The C5 nerve roots were found to exit over the middle aspect of the intervertebral disc, whereas the C6 and C7 nerve roots were found to traverse the proximal part of the disc. The C8 nerve roots had little overlap with the C7–T1 disc in the intervertebral foramen. The C6 and C7 rootlets passed two disc levels in the dural sac. Also, a high incidence of the intradural connections between the dorsal rootlets of C5, C6, and C7 segments was found. Conclusions. This study demonstrated the anatomy of the nerve roots, rootlets, and intervertebral foramina, and may aid in understanding the pathology of cervical radiculopathy. The presence of intradural connections between dorsal nerve roots and the relation between the course of the nerve root and the intervertebral disc may explain the clinical variation of symptoms resulting fromnerve root compression in the cervical spine. To perform cervical foraminotomy for cervical radiculopathy, it is necessary to understand the detailed anatomy of the intervertebral foramina thoroughly.

Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart

SummaryThe role of actin bundles on the heart looping of chick embryos was examined by using cytochalasin B, which binds to the barbed end of actin filaments and inhibits association of the subunits. It was applied to embryos cultured according to News method. Looping did not occur when cytochalasin B was applied diffusely in the medium. Further, we disorganized actin bundles in a limited part of the heart tube to examine the role of actin bundles in each part in asymmetry formation. A small crystal of cytochalasin B was applied to the caudal part of the heart tube on either the left or right side. The disorganization of actin bundles on the left side resulted in the right-bending of the heart, an initial sign of dextro-looping (normal pattern), and right side disorganization resulted in left-bending. We suggest that actin bundles on the right side of the caudal part of a heart tube generate tension and cause dextro-looping. Embryos whose hearts bent to the right rotated their heads to the right, and embryos with left-bent-hearts rotated their heads to the left. The rotation of the heart tube may therefore decide in which direction the body axis rotates.

Disturbance of refinement of retinotectal projection in chick embryos by tetrodotoxin and grayanotoxin

The role of neuronal activity in the refinement of the retinotectal projection in chick embryos was studied using tetrodotoxin (TTX) which blocks sodium channels and grayanotoxin I (GTX) which opens the channels. Optic nerve fibers were traced by the fluorescent dye DiI. The toxins were injected into the eyeball during the refinement phase of retinotectal projection (either the 13th, 14th or 15th day of incubation). A tiny crystal of DiI was placed in the temporal part of the retina. In the control embryos, fibers caudally overshooting and arborizations outside the terminal zone were found before maturation of retinotectal projection. Overshooting fibers regressed linearly, and aberrant arborizations reduced quadratically, and then the precise retinotopic map is formed by stage 44 (18th day of incubation). Both TTX and GTX interfered with the regression of overshooting fibers and arborizations outside the terminal zone. The initial action of the toxins are reverse, but their final effect may be comparable, and they may interfere with synchronous firing of the neighboring fibers. Our results emphasize a role of neuronal activity in the refinement of retinotectal projection.

Bone-Staining Technique for Fetal Rat Specimens without Skinning and Removing Adipose Tissue

Abstract: A rapid procedure was developed for staining fetal rat specimens. Full‐term rat fetuses were fixed in 99% ethanol and only thoracoabdominal organs were removed. Without skinning and removing adipose tissue and eye balls, the specimens were placed in 85% ethanol containing 1.5% KOH and 0.0015% alizarin red S for 5 days. The specimens were macerated in 1% aqueous KOH and cleared in 50% glycerin solution. The skeleton was visible through surrounding tissue containing adipose tissue which became transparent.

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum.

Abstract: Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2‐4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5‐bromo‐2′‐deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5‐trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.

Changes in the arrangement of actin bundles during heart looping in the chick embryo

SummaryWe assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.

Pathogenesis of cleft palate in mouse embryos exposed to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate in mouse embryos. It has been believed that TCDD inhibits palatal fusion by suppression of disappearance of medial edge epithelial (MEE) cells on palatal shelves. However, we found that exencephalic mouse embryos were resistant to the cleft palate-inducing action of TCDD. In the present study, we examined cell kinetics in MEE and palatal mesenchyme in embryos exposed to TCDD with or without exencephaly for elucidation of pathogenesis of cleft palate by TCDD. Pregnant Jcl:ICR mice were given TCDD orally at 40 microg/kg at gestation day (GD) 12.5. Embryos were harvested between GD 13.5 and GD 14.5 and examined for cell kinetics by bromodeoxyuridine (BrdU) and TUNEL methods. Exencephaly was induced by intraperitoneal injection of CdCl(2) at 6 mg/kg at GD 7.5. BrdU-positive cells were decreased in TCDD-treated embryos in MEE and mesenchymal cells. TUNEL-positive cells were detected in MEE both in TCDD-treated and untreated control embryos, as well as in embryos with or without exencephaly. We also measured the gap between shelves between GD 14. 0 and GD 14.5. There were no differences at GD 14.0 between control and TCDD-exposed embryos, but at GD 14.25 and GD 14.5, TCDD-exposed embryos had wider gaps than controls. These findings indicate that cleft palate by TCDD results from poor development of palatal shelves. Teratogenesis Carcinog. Mutagen. 20:73-86, 2000.

Neurotoxic effects of phenytoin on postnatal mouse brain development following neonatal administration.

Phenytoin (PHT) is a commonly used anticonvulsant drug. It has been reported that children exposed prenatally to PHT have brain malformations and psychomotor dysfunction. The neonatal development of the central nervous system (CNS) in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT on postnatal brain development, we administered PHT at doses of 10, 17.5, 25, or 35 mg/kg to newborn mice once a day during postnatal days (PD) 2-4. These dose levels result in plasma levels corresponding to the therapeutic ranges in humans. We measured the weight of total brain, cerebrum, cerebellum, and brain stem on PD 5 through 21, and examined early motor functions including head elevation, elevation of pelvis, pivoting, crawling, and righting reflex . Total brain weight, cerebral weight, and cerebellar weight in the group treated with 25 or 35 mg/kg were significantly reduced compared to controls from PD 5 to 21. Mice treated with PHT at 25 or 35 mg/kg showed decreased locomotor abilities and righting reflex on PD 5. In all phenytoin treatment groups, phenytoin levels in the brain were higher than those in the plasma on the third day of PHT treatment. We thus observed neurotoxic effects of PHT on postnatal brain development in mice. Our present data may provide useful implications for the management of PHT-induced developmental neurotoxicity and evaluation of psychomotor development in children exposed to PHT during the late fetal period.