Twin-twin transfusion syndrome in a dizygotic monochorionic-diamniotic twin pregnancy.

We present a case of twin-twin transfusion syndrome with discordant gender. Monochorionicity was confirmed by surgical pathology. Cytogenetic analysis showed normal 46,XX and 46,XY karyotypes. Microsatellite analysis using reliable pericentromeric markers was consistent with dispermic fertilization of two separate ova. This suggests that monochorionicity, rather than zygosity, may be responsible for the development of placental vascular anastomoses.

Differences in microcirculatory bed arrangement in some skeletal muscles of the rat and golden hamster.

The architectural features of the most peripheral parts of the microcirculatory bed were studied in selected skeletal muscles (cremaster, retractor, spinotrapezius, and ventral stripe of spinotrapezius) in rats and golden hamsters ranging in weight between 130 and 330 and 130 and 180 g, respectively. In the cremaster muscle of both species, a very complicated, irregular and heterogenous arrangement of terminal arterioles, as well as of postcapillary and collecting venules was found. In the retractor, the most regular arrangement was along the margins of the middle part of muscle, but an irregular brush-like branching of the terminal arterioles and collecting venules limits the use of these areas. Similarly, in spinotrapezius muscle the most regular vascular architecture, complicated by a different fashion of terminal arterioles and collecting venules, was along the ventral border of muscle. The most regular and simplest vascular arrangement was in the ventral stripe of spinotrapezius of the rat. Therefore, this muscle is recommended as most appropriate for intravital experimental studies of the skeletal muscle microcirculatory bed in all age and weight groups.

Induction of microsomal epoxide hydrolase activity in inbred mice by chronic phenytoin exposure.

A lower microsomal epoxide hydrolase (mEH) activity has been associated with increased likelihood of fetal hydantoin syndrome. While phenytoin anticonvulsive regimens are long-term, there are no data regarding induction of mEH by chronic phenytoin exposure. Two inbred mouse strains which differ in their susceptibility (A/J > C57BL/6J) to phenytoin-induced oral clefting were treated with an oral gavage of phenytoin for 14 consecutive days. The mice were sacrificed on the 15th day, and hepatic microsomes were prepared. mEH activity was determined using benzo[a]pyrene-4,5-oxide. The dihydrodiol product was separated by HPLC and quantified. There was no significant difference (P = 0.15) in the phenytoin plasma level between the two strains on Day 15. There was no significant difference (P = 0.07) between control and sham control groups within each strain, so they were combined for further analysis. There was a significant strain difference (P = 0.0001) between the control and phenytoin-exposed group means, with the C57BL/6J strain having the greater activity before and after phenytoin exposure. The A/J phenytoin-exposed group activity was 51% higher (P = 0.01) than the A/J control, while the C57BL/6J phenytoin-exposed group activity was 78% higher (P = 0.001) than the C57BL/6J control. The greater mEH activity in the phenytoin-induced clefting resistant strain (C57BL/6J) before and after phenytoin exposure is consistent with a putative oxidative metabolism mechanism of phenytoin teratogenecity. Chronic phenytoin exposure induced mEH activity in both strains, although the strain with the greater enzyme activity prior to the exposure continued to have the greater activity following induction.

Basement membrane composition of cartilage canals during development and ossification of the epiphysis.

BACKGROUND: Cartilage canals are perichondral invaginations of blood vessels and connective tissue that are found within the epiphyses of most mammalian long bones. Functionally, they provide a means of transport of nutrients to the hyaline cartilage, a mechanism for removal of metabolic wastes, and a conduit for stem cells that are capable of initiating and sustaining ossification of the chondroepiphysis. Morphological and biomolecular changes of the chondroepiphyses appear to potentiate ossification within the chondroepiphyses of developing bones. METHODS: As both cell migration and vascular invasion are anchorage dependent processes, antibodies to laminin and Type IV collagen were used to assess compositional changes in the basement membrane of cartilage canals accompanying epiphyseal ossification. RESULTS: Differences in chronological appearance, as well as, in distribution between the two components were noted in the chondroepiphysis. Laminin was distributed throughout the connective tissue of cartilage canal at all stages of development, and not limited to an association with the vascular lumen. Type IV collagen was not present during the initial perichondral invagination. Although staining for Type IV collagen was later acquired, its distribution was restricted to a discontinuous rimming of the periphery of the canal, and a diffuse presence within the intra-canalicular mesenchyme. CONCLUSIONS: Concurrent with chondrocyte hypertrophy and mineralization of the hyaline matrix, rapid changes in both the morphology of the vessel and distribution of the antibodies were detected. In addition to the presence of laminin at the interface of the endothelium and the hyaline matrix, a wide distribution within the connective tissue components of the newly ossifying matrix of epiphyseal bone could be detected. Type IV collagen remained closely associated with the lumens of the intra-canalicular vessels throughout the transition. Following ossification of the secondary center, staining for Type IV collagen could then be detected in the bone-forming regions of transforming matrix as well, clearly delineating the individual vessels within the newly formed marrow spaces. This suggests that bone formation is intimately related to vessel staining for collagen type IV, and that acquired vessel competence is a facet of endochondral bone formation that results from provisional matrix changes. Furthermore, the data suggests that during bone formation under tension, basement membrane deposition can be demonstrated without an intermediary hyaline matrix hypertrophic chondrocyte phase. This data was interpreted to suggest that chondrocyte hypertrophy at the growth plate may be a reaction to vascular invasion, that in turn, stimulates adjacent chondrocyte proliferation.

Adherence of Treponema pallidum subsp. pallidum in the rabbit placenta.

Congenital syphilis is the consequence of transplacental passage of Treponema pallidum. A system was developed to deliver virulent T. pallidum, Nichols strain, through an isolated uterine horn of a pregnant rabbit in order to investigate the mechanism by which T. pallidum is able to cross the placenta. While the pregnant rabbit was anesthetized, the ovarian artery and the uterine vein were cannulated and attached to a peristaltic pump. Treponema pallidum (2-5 x 10(8) in 10-15 ml RPMI-1640) were circulated via the peristaltic pump throughout the horn for 2 hr, after which the placentas were removed, fixed in formalin, and embedded in paraffin. This system was used to investigate treponemal binding to rabbit placenta at Day 20 and 26 during the gestation period of the rabbit (29-32 days). Examination of 5-microns Dieterle silver stained tissue sections revealed (i) a greater number of spirochetes in the later gestational stage placentas (Day 26) than in the earlier placentas (Day 20), (ii) organisms adhering to the trophoblastic tissue surrounding the maternal blood channels, and (iii) organisms appearing to be in the process of penetrating the trophoblastic tissue or that had completely penetrated from the channels into the trophoblastic elements. We suggest that T. pallidum may be adhering to placental components that are differentially expressed during gestation of the rabbit.

Lipoxygenase activity in rat embryos and its potential for xenobiotic oxidation.

This paper reports dioxygenase activity in rat embryos and demonstrates for the first time the ability of rat embryo lipoxygenase (LO) to oxidize xenobiotics in vitro. Significant dioxygenase activity towards linoleic acid was found in cytosol isolated from rat embryos in different developmental stages on day 9 and 10 of gestation. All four xenobiotics tested were oxidized at significant rate by the LO in the presence of linoleic acid. Both dioxygenase activity and benzidine oxidation were inhibited by the known inhibitors of LO, i.e. nordihydroguaiaretic acid and eicosatriynoic acid. These findings suggest that LO may be an important pathway for xenobiotic metabolism in the rat embryo.

Rat hepatic glutathione S-transferase-mediated embryotoxic bioactivation of ethylene dibromide.

The embryotoxic effects of ethylene dibromide (EDB) bioactivation, mediated by purified rat liver glutathione S-transferases (GST), were investigated using rat embryos in culture. Significant EDB metabolism was observed with rat liver GST purified by affinity chromatography (specific activity of 188 +/- 11.3 nmol/min/mg protein). The reaction was enzymatic in nature and the conjugation rate was proportional to the concentration of EDB (up to 0.75 mM) and the enzyme present in the reaction medium. EDB activation by 100 units (1 unit = 1 nmol of glutathione consumed per min) of purified rat liver GST caused a significant reduction in general development as measured by crown-rump length, yolk sac diameter, somite number, and the composite score for different morphological parameters (Brown and Fabro methodology). Structures most significantly affected were the central nervous and olfactory systems as well as the yolk sac circulation and allantois. The results of this study clearly indicate that under in vitro conditions, bioactivation of EDB by GST can lead to embryotoxicity.

Statistical analysis of the effect of cadmium and zinc on hamster teratogenesis.

Maternal smoking is correlated with lower average birth weights and an increase in malformations in some studies. Increased maternal cadmium levels and reduction of zinc levels in certain tissues from fetuses of women who smoke suggest a biological association during pregnancy. Zinc has a protective effect on hamster teratogenesis caused by cadmium. To determine whether this protective effect is additive or synergistic (interactive), pregnant golden Syrian hamsters were injected (iv) on Day 8 of gestation with a test solution based on maternal body weight (0.5 ml per 100 g). Five doses were given: 2 mg/kg zinc chloride, 2 mg/kg cadmium chloride, 3 mg/kg cadmium chloride, 2 mg/kg zinc chloride plus 2 mg/kg cadmium chloride, and 2 mg/kg zinc chloride plus 3 mg/kg cadmium chloride. Fifty dams were randomly placed into one of the groups, for a total of 10 pregnant dams in each group. Twenty other dams were randomly placed into untreated or saline control groups. Fetuses were recovered on Day 15 and weighed, crown-rump length was measured, and fetuses were examined for viability and external malformations. Resorptions were noted. Statistical analyses included one- and two-way nested ANOVA, and logistic regression adjusted for litter effect. Zinc's protective effect on acute cadmium embryonic/fetal toxicity and teratogenicity was confirmed. The protective effect was of the same magnitude relative to the dose-dependent effect of the cadmium exposure, indicating that the effect was statistically additive and not synergistic. This suggests that the effect depends on competition of the two elements at some common binding site(s).

A novel model to assess developmental toxicity of dihaloalkanes in humans: bioactivation of 1,2-dibromoethane by the isozymes of human fetal liver glutathione S-transferase.

Glutathione S-transferase (GST) isozymes from human fetal liver (16-18 weeks gestation) were purified by affinity chromatography followed by ion-exchange high performance liquid chromatography (HPLC). The purified isozymes were used to investigate toxicity of 1,2-dibromoethane(EDB) in an in vitro model of rat embryos in culture as passive targets. At least five isozymes of GST were found in the human fetal liver. Two anionic forms [pI values 5.5 (P-2) and 4.5 (P-3)] and one basic form [pI value 8.7 (P-6)] were clearly separated. The presence of two near-neutral forms was also identified. All the isozymes of the human fetal liver GSTs tested metabolized EDB (specific activities were 2.1, 7.0, and 2.0 mumol of GSH consumed/min/mg protein for P-2, P-3, and P-6 isozymes, respectively). Covalent binding of EDB to DNA and protein was 144% and 212% higher, respectively, with the P-3 anionic isozyme when compared to the P-6 basic isozyme of GST. No covalent binding to either protein or DNA was observed with the P-2 isozyme. EDB bioactivation by the GST isozyme P-3 (15 units; 1 unit = 1 nmol of GSH consumed/min) resulted in toxicity to cultured rat embryos. Significant reductions of crown rump length, yolk sac diameter, and the composite score of morphological parameters (Brown and Fabro method) were observed. The central nervous system, optic and olfactory systems, and the hind limb were most significantly affected. The results of this investigation suggest that EDB may be classified as a suspected developmental toxicant in humans.

Computed analyses of the pathomechanics of scaphoid waist nonunions.

The computed tomography scans of both the injured and the normal wrists of nine patients with a scaphoid nonunion of 5 to 120 months' duration were used to create three-dimensional computer models. When the computer images of the normal and the contralateral fractured scaphoids were superimposed, it was possible to calculate the volume of bone that was lost as a result of the injury and its failure to heal, as well as the angular relationship of the fracture components to one another. The amount of the scaphoid bone that was lost varied from 6% to 15% of bone volume and did not show a linear correlation with the duration of the nonunion. The configuration of the missing bone was consistent and exhibited a prismatic shape whose base is quadrilateral and faces palmarly. The proximal scaphoid fracture component is extended, radially deviated, and supinated in relation to its distal fracture component. The consistent fracture deformity and the configuration of the bony defect in the scaphoid waist nonunions should be helpful in the understanding and treatment of the condition.

Regional changes in hemodynamics and cardiac myocyte size in rats with aortocaval fistulas. 2. Long-term effects.

Regional changes in hemodynamics and cardiac myocyte size were examined in adult rats 5 months after creating a large aortocaval fistula. At that time, cardiac output, left and right ventricular pressures, and left and right ventricular dP/dtmax were measured. Subsequently, isolated cardiac myocytes were collected from the left ventricle, right ventricle, and septum for cell size measurements. Compared with sham-operated controls, percent dry weight was reduced in the liver and kidney but was unchanged in the lung. Heart rate, left ventricular systolic pressure, left ventricular dP/dtmax, and systolic aortic pressure were not changed in rats with fistulas. However, cardiac output, stroke volume, left ventricular end-diastolic pressure, and all measured parameters in the right ventricle were significantly increased. Mean cell volume and the ratio of heart weight to body weight were both elevated 92%. Cell volume, cell length, and cross-sectional area increased significantly in each heart region examined. Hypertrophy was more pronounced in cells from the right ventricle and the endomyocardium of the left ventricle. The percentage of cells with mononucleation or binucleation was not changed in any heart region of rats with fistulas. In summary, despite evidence of renal and hepatic congestion, most indexes of cardiac function were normal or elevated 5 months after creation of a large volume-overload-induced hypertrophy. Data from isolated cardiac myocytes suggested that cellular hypertrophy, rather than hyperplasia, was responsible for the increased cardiac mass.

Regional changes in hemodynamics and cardiac myocyte size in rats with aortocaval fistulas. 1. Developing and established hypertrophy.

The effects of a large arteriovenous fistula on left and right ventricular hemodynamics and cardiac myocyte size were examined in adult rats at 1 week and 1 month after surgery. Cardiac output, left ventricular function, and right ventricular function were evaluated before obtaining isolated myocytes for cell size measurements. Average heart weight increased 35% at 1 week and 86% at 1 month in rats with fistulas. In general, myocyte hypertrophy was due to a proportional increase in length and width (length/width ratio remained constant). This change was more evident in the large hearts from rats with 1-month fistulas. At both the 1-week and 1-month intervals, the hypertrophic response of right ventricular myocytes was slightly greater than that observed in the left ventricle or interventricular septum. Left ventricular systolic pressure and dP/dtmax were significantly reduced at 1 week but returned to normal after 1 month of overloading. Left ventricular end-diastolic pressure was increased approximately fivefold and twofold at 1 week and 1 month, respectively. Right ventricular systolic pressure and dP/dtmax were increased at both intervals examined. We conclude that severe volume overloading from a large aortocaval fistula in the rat is characterized by 1) depressed left ventricular function at 1 week followed by a large compensatory hypertrophy and near normal function at 1 month, 2) right ventricular pressure overload, and 3) changes in myocyte shape that resemble normal physiological growth.

Carpal orientation from computed reference axes.

Carpal instability is usually diagnosed by abnormal two-plane radiographic angles. These angles are often unreliable. A method that eliminates interpretation of overlapping shadows and uses all of the carpal geometry should improve clinical diagnoses. The digital data from computed tomography scans can be manipulated to describe the carpal orientation in the normal wrist. The digital data from the computed tomography scans of twenty-two normal wrists were used to compute distances with and without directions between the volumetric centroids of the carpal bones. An expansion technique also extracted from the computed tomography data an orthogonal set of vectors, the principal axes. The first principal axis describes the longest dimension of each bone. The average angle produced by the first principal axes of the scaphoid and lunate was 23.6 degrees, scaphoid and capitate was 73.2 degrees, and the capitate and the lunate was 93.5 degrees. These computations represent new carpal axes and intercarpal angles that are not related to the commonly measured two-plane radiographic angles. They should prove helpful in the study of kinematics and pathomechanics in the wrist joint.

Structural remodeling of cardiac myocytes in rats with arteriovenous fistulas.

Structural changes in the heart associated with chronic volume overloading have not been adequately documented. In anesthetized adult rats, microsurgical techniques were used to produce two types of arteriovenous shunts. The end of a femoral artery was connected to the side of a femoral vein to produce a femoral shunt. Aortocaval fistulas were produced by placing a venous graft (femoral vein) between the abdominal aorta and inferior vena cava. A polyethylene ring was placed around the venous graft to control the size of the shunt. After 10 weeks, rats were anesthetized and hearts were excised, weighed, and perfused with collagenase to obtain isolated myocytes. Heart weight/body weight of rats increased 15% (p less than 0.005) with unilateral femoral fistulas and 41% (p less than 0.001) with aortocaval fistulas. Length of isolated cardiac myocytes was measured directly with a microscope. Cell volume was measured with a Coulter Channelyzer. Myocyte cross-sectional area was calculated from cell volume/cell length. Cell volume increased in proportion to heart weight in each experimental model. With both types of shunts, the majority of myocyte hypertrophy was due to an increase in cell length. There was a trend toward an increase in cross-sectional area in each region of both fistula groups, although this change was not statistically significant. These results indicate that eccentric cardiac hypertrophy was associated with enlargement of individual myocytes primarily as a result of increased length.

Mathematical analysis of computed carpal models.

The computed carpal models from digital computed tomography (CT) data obtained in this study compare favorably to natural anatomy. A new application of algebraic analysis of this data provides mathematical markers from which to calculate the position and orientation of each carpal bone. When the origin of the spatial coordinates of a carpal bone is transferred to the centroid of the bone, the data can be treated as three-dimensional pattern vectors describing its surface. It is then possible to calculate vectors that are the principal axes of the geometry. These axes provided references that were used to calculate position and orientation of the carpal bones in three wrist specimens. Comparisons of volumes, surface areas, and sizes and proportions of five computed images of each carpal bone from two of these wrists demonstrate the reliability of the technique. The analysis of CT scans of ceramics with known boundaries allows an estimation of its accuracy. The technique is well suited to the future study of normal wrist kinematics and pathological conditions.

Scaphoid orientation and location from computed, three-dimensional carpal models.

Subtle subluxations within the carpus are often difficult to diagnose. Carpal orientation and location can now be quantified by mathematical analysis of computed, three-dimensional models produced from serial CT scans. The technique and its application in the analysis of scaphoid subluxation are described.

Sirenomelia: analysis in the cadmium- and lead-treated golden hamster.

Sirenomelia, a fusion of the lower extremities, is believed to result from a median, bilateral symmetric defect of the caudal portion of the embryo at a very early stage in development. Anomalies of the gastrointestinal and urogenital systems are commonly associated with this malformation. Sirenomelia is not an embryo-lethal condition but typically is incompatible with postnatal life when combined with the associated malformations. In this study, intravenous treatment of hamsters with a combination of cadmium and lead on the morning of the eighth day of gestation resulted in 1.4, 22.2, and 35.6% of the viable fetuses displaying sirenomelia in litters recovered on days 15, 12, and 10 of gestation, respectively. With the exception of several fetuses with exencephaly, most structures cranial to the level of the umbilicus were normal. Caudal abdominal and pelvic structures were severely affected, with agenesis or dysgenesis of the kidneys, bladder, umbilical arteries, and external genitalia frequently noted. The administration of a combination of cadmium and lead has proven to be an effective and consistent means of inducing sirenomelia in the golden hamster.

Endotoxin-induced hyperthermia in pregnant golden hamsters.

The degree of elevation and the duration of induced fever following endotoxin treatment of pregnant golden hamsters were examined. A radiothermistor was surgically implanted in the abdomen of each animal on day 6 of pregnancy. The animals were intravenously treated with a bolus injection of saline or endotoxin (1, 10, 20, or 200 micrograms/kg) on the morning of day 8 of gestation. Abdominal temperatures were monitored at 15-minute intervals on days 8 and 9 and at 1-hour intervals on days 10 through 14 of gestation. The mean temperature of the saline-treated control animals was 37.2 degrees C with a range from 36.5 to 38.2 degrees C. The total number of viable fetuses, and the mean fetal weight of the 1-microgram/kg group were not significantly different from those of the saline controls, although a statistically significant monophasic temperature elevation did occur between 3 and 9 hr post-treatment. Three of seven females that received 10 micrograms/kg had viable litters and exhibited a monophasic fever. The remaining four animals exhibited a biphasic fever and total resorption of all implanted fetuses. The first phase began 2.5 hr post-treatment and lasted for 3-6 hr. The second phase began 12-15 hr post-treatment and lasted for 15 to 25 hr. All animals receiving 20 or 200 micrograms/kg demonstrated similar multiple temperature elevations. Treatment of animals with greater than 20 micrograms/kg resulted in total resorption of all implanted fetuses and maternal weight loss. Nonpregnant females treated with 20 micrograms/kg of endotoxin exhibited a biphasic temperature elevation; thus the second phase of the fever response can not be attributed to embryo death and resorption.

Effects of endotoxin on placental labyrinth formation in the golden hamster: a light and electron microscopic study.

Animal studies of endotoxemia during pregnancy have suggested that the placenta is the primary site of action. This study was designed to evaluate the effects of endotoxin at previously established resorptive and teratogenic dose levels during the development of the placenta in the hamster. At the higher dose levels, total necrosis of the placenta was observed within 24 hr after treatment. Formation of fibrin thrombi was only apparent well after the initiation of necrotic events. At the teratogenic dose, a marked decrease in the formation of the placental labyrinth was evident, but invading fetal capillaries and fetal blood cells appeared normal. However, scattered necrotic foci of trophoblastic cells were observed.

Teratogenic effects of endotoxin on the golden hamster.

Gram-negative urinary tract infections in pregnant women have been implicated as causes of maternal endotoxemia and a subsequent higher incidence of malformations in their offspring. A study was performed to evaluate the effects of endotoxin on the development of the golden hamster. Endotoxin was shown to be extremely embryolethal at higher doses and to produce several malformations at lower doses. The pregnant hamster and its developing embryos were observed to be far more sensitive to endotoxin than species examined by other investigators.