Cleft palate and open eyelids inducing activity of lorazepam and the effect of flumazenil, the benzodiazepine antagonist.

Lorazepam (Ativan, Wyeth) at dosages of 20-36 mg/kg was used to test for developmental toxicity in the mouse embryo/foetus model. Two separate regions were considered: (1) the central nervous system and (2) the roof of the mouth and the eyelids. In the first case a single administration of lorazepam was applied at the very beginning of the 9th gestation day. In the second, it was administered in preliminary tests on two consecutive gestation days between the 11th and 14th days and in later experiments once only on the 13th or 14th gestation day. In the first part of investigations regarding the development of the central nervous system, lorazepam unlike many other neurotropic drugs, was found not to induce any aberrations in the process of the neural tube closure. In the second part, in which palate closure and the temporary closure of eyelids were monitored, it was found that lorazepam does interfere with these processes. In order to test whether lorazepam's neurocristopathic activity can be prevented, suggesting the presence of benzodiazepine receptors in the neural crest cells, we used the benzodiazepine antagonist, flumazenil (Anexate, Roche). The results of these experiments indicated the flumazenil was able to prevent cleft palate and open eyelids cases almost completely if it was administered 3 hr after administration of lorazepam. If the treatments were administered in the reverse order, the frequency of neurocristopathy cases was unaffected, i.e. flumazenil did not influence the teratogenic activity of lorazepam.

The absence of teratogenic effects of some analgesics used in anaesthesia. Additional evidence from a mouse model.

The possibility exists that agents used in anaesthesia may have adverse teratogenic effects on staff, patients, and developing fetuses. It has been shown that a range of neurotropic drugs, when injected into pregnant mice on the 9th day of gestation, produce a characteristic group of central nervous system malformations in their fetuses. We have studied the possible teratogenicity of pethidine, fentanyl, phenoperidine and lignocaine when tested in this way and conclude that they appear to have less effect than other neurotropic drugs previously tested.

Teratogenic potential of two neurotropic drugs, haloperidol and dextromoramide, tested on mouse embryos.

Potential teratogenic activity of two neurotropic drugs, haloperidol and dextromoramide tartrate, was tested by using the mouse embryo experimental model. Like numerous other drugs of this class these two are also embryotoxic. After treatment 1 hour into the 9th gestation day they induce the neurotropic syndrome of malformations comprising exencephaly, craniorachischisis, kinking of the spinal cord, brachyury, and dilation of the fourth brain ventricle. In addition, dextromoramide tartrate was found to induce one more, so far unknown, neural tube defect, namely the ectopia of the neural tube. Delay of treatment by 1 or 2 hours tends to displace the location of the neural tube defects along the length of the neural axis towards the anterior and posterior directions. Even after accumulating these results at relatively high doses, it is difficult to estimate human reproductive risks from this animal data for human therapeutic doses.

Teratogenic activity of lithium carbonate: an experimental update.

The teratogenic potential of lithium carbonate was re-examined using mouse embryos as an experimental model. Externally it was found that, like many other psychoactive drugs, it exhibits a teratogenic activity that is confined to the development of the central nervous system. When administered intraperitoneally to pregnant females in doses ranging from 330 to 340 mg/kg at the very beginning of the ninth gestation day, i.e., during the critical period of the neural tube closure, it is embryotoxic, causes retardation of development, and induces the "neurotropic syndrome of malformations" comprising exencephaly, craniorachischisis, rachischisis, kinking of the spinal cord, and dilation of the fourth brain ventricle. Depending on the exact time of gestation when lithium carbonate is administered, the frequencies of exencephaly and spinal kinking after treatment 1 hour into the ninth day are compared with those after treatment 2 or 3 hours into the ninth day, incidence of exencephaly is increased at the later time and the incidence of spinal kinking is decreased.

The interference of naloxone hydrochloride in the teratogenic activity of opiates.

Diamorphine hydrochloride, methadone hydrochloride, and the synthetic enkephalin analogue FK 33-824 are potent teratogens for the central nervous system in mouse embryos. They induce the "neurotropic syndrome of malformations," which is restricted to the central nervous system if administered during the critical period of neural tube closure. Pretreatment with corresponding equimolecular doses of the antagonist naloxone hydrochloride applied 30 minutes before treatment with the opiate agonists abolishes the major severe malformations, i.e., exencephaly, craniorachischisis, and brachyury, and reduces the number of cases of kinking of the spinal cord. Dilation of the fourth brain ventricle remains unaffected. It is suggested that the mechanism of interference in the teratogenicity of the opiates by naloxone hydrochloride reported here is based on competition for opiate receptors. In general, these observations are regarded as evidence that the pharmacological affinity of opiate agonists to receptors in the central nervous system is responsible for the malformations caused by them in this system.

Experimental analyses of cytoplasmic rearrangements which follow fertilization and accompany symmetrization of inverted Xenopus eggs.

Cytoplasmic rearrangements which follow fertilization were monitored in inverted eggs. A set of yolk compartments was resolved by cytological analyses of both normally oriented and inverted eggs. Those compartments were characterized by their yolk platelet compositions and movement during egg inversion. In addition to the major yolk masses which contain either small, intermediate or large platelets, minor cytoplasmic compartments which line the egg cortex were also identified. During egg inversion the yolk compartments shift. Those yolk mass shifts occurred only after the inverted egg was activated (by sperm, electrical or cold shock). The direction of shift of the major yolk components, rather than the sperm entrance site (as in normal orientation eggs), determines the dorsal/ventral polarity of the inverted egg. Among different spawnings the rate of shift varied. Eggs that displayed the fastest rate of shift exhibited the highest frequency of developmental abnormalities during organogenesis. Isopycnic density gradient analysis of yolk platelets and blastula blastomeres showed that isolated yolk platelets and mid-blastula blastomeres are not of uniform buoyant density. Three major yolk platelet density bands were resolved. Large, intermediate, and small yolk platelets were found in all bands. The high density band had the largest proportion of the large yolk platelets and the low density fraction showed the largest proportion of the small yolk platelets. Interpretation of novel observations on cytoplasmic organization provided criticisms of some earlier models. A new 'Density Compartment Model' was developed and presented as a coherent way to view the organization of the egg cytoplasm and the development of bilateral symmetry.

Pattern formation in amphibian embryos prevented from undergoing the classical "rotation response" to egg activation.

Fertile Xenopus laevis eggs were immobilized so that they were prevented from undergoing the "rotation response" to activation. Many of those unrotated eggs developed through organogenesis, indicating that egg rotation is not a prerequisite for normal early embryogenesis. Various aspects of the regulation of pattern formation were analyzed in unrotated eggs: It was discovered that a substantial rearrangement of yolk platelets occurred without affecting subsequent pattern formation. The germ plasm, however, remained localized in the vegetal hemisphere in inverted eggs. Cleavage furrows and the site of involution were both often observed in novel locations in inverted eggs which were prevented from rotating during activation.

Tissue interactions during axial structure pattern formation in amphibia.

Tissue interactions have traditionally been assigned important roles in establishing the pattern of amphibian axial structure morphogenesis. Those interactions have been postulated to generate the patterns of neural fold morphogenesis, neural tube formation, and somite development. A review of axial structure development together with a brief discussion of the classical viewpoint, is presented. A re-examination of axis formation has recently been carried out with the SEM. Embryos which displayed major defects in notochord development, ranging from diminished length to complete obliteration, were produced by irradiating fertile eggs prior to first cleavage. A comparative SEM analysis of normal and "notochord defective" embryos revealed that, contrary to previous reports, the notochord is apparently a dispensable component of the developing axial structure system. Lastly, TEM examination of the notochord defective embryos allowed some insight into the ultrastructural alterations which occur in the notochord and neural tube cells of irradiated embryos. Additional information about the structure of the notochord, and the cellular mechanics of somitogenesis emerged from those studies.

MALFORMATIONS OF THE CENTRAL NERVOUS SYSTEM INDUCED BY NEUROTROPIC DRUGS IN MOUSE EMBRYOS.

Out of a sample of fifteen neurotropic drugs consisting of seven antidepressants and anti-psychotics, two antianxiety drugs, one anticonvulsant, three opiates and two synthetic analgesics, twelve were found to be teratogenic for mouse embryos, causing malformations of the central nervous system. After single injections of the teratogenic dose administered at the very beginning of the ninth day of gestation, four days later, i.e. in 13-day-old embryos, the induced defects appeared to make up a recurring syndrome of malformations which consists of several abnormalities present in various frequencies either individually or in combination in the same embryos. These malformations are: exencephaly, craniorachischisis, cervical and thoraco-lumbar myeloschisis, hydrocephalic dilatation of the fourth brain ventricle, Z-shaped kinking of the spinal cord and lumbar hydromyelia. In addition, after administration of some of the drugs, branchyury or anury with or without lumbar myeloaplasia were recorded. In general the results reported here seem to suggest that because of their possible affinity neurotropic drugs are potentially teratogenic for the embryonic central nervous system if applied at the time of the neural tube closure although it is known that there are drugs in this group which do not cause any malformations of the central nervous system and that many non-neurotropic agents do cause such malformations. Secondly, the results seem to suggest also that the position of the malformations along the cerebro-spinal axis may be depending to some extent on the pharmacological properties of the drugs tested. These conjectures are treated here as entirely provisional pending further investigations.

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.

Electron microscopic studies on chick limb cartilage differentiated in tissue culture.

The hind limb-bud mesenchyme of chick embryos 4-4 1/2 days old was cultured in Eagle's Minimum Essential Medium supplemented with both horse serum and fresh chick embryo extract. Whereas no differences are seen at the light-microscope level, at the electron-microscope level the chondroblasts differentiated in tissue culture are noticeably different from those differentiated in vivo, particularly in the possession of some cytoplasmic fibrils and vacuoles. It is proposed that the secretion of the extracellular matrix alone is not sufficient to account for the pattern of cellular arrangement in a cartilaginous condensation.

Ultrastructural aspects of histolytic processes in the salivary gland cells during metamorphic stages in Rhynchosciara hollaenderi (Diptera, Sciaridae).

The cytoplasm of Rhynchosciara hollaenderi late larval, prepupal and pupal salivary gland cells was studied at the ultrastructural level. In the second half of the 4th instar, evidence of an intensive secretory activity is visible in the form of numerous secretory granules in the apical area of the cells. At the same stage, the endoplasmic reticulum cisternae adjacent to Golgi groups are active in the transfer of vesicular elements. At later stages this activity rapidly diminishes. Before the appearance of the DNA puffs, i.e. at the end of the 4th instar, mitochondria begin to show a granular deposit and normal mitochondria decrease in number. These with the granular deposit form clusters and initiate formation of single autophagic vacuoles before the appearance of the DNA puffs. Later, at the time, when the 2B puff opens, the autophagic vacuoles appear in great number. Simultaneously with the formation of the autophagic vacuoles the presence of acid phosphatase in the Golgi vesicles and in autophagic vacuoles was shown. In the last stages investigated (late pupae) acid phosphatase is present free in the cytoplasm and at the same time disappearance of free ribosomes, pycnosis of polytene chromosomes and breakage of nuclear membranes occur. It is concluded that the histolysis of the salivary gland cells begins before the large DNA puffs appear, then it becomes very intensive and continues after these puffs undergo regression.