Spatial pattern analysis of nitrergic neurons in the developing myenteric plexus of the human fetal intestine.

BACKGROUND: Enteric nervous system precursors derived from the neural crest migrate along defined pathways to colonize the bowel. The individual cells in different environments experience different growth, differentiation, and survival conditions. Hence, the spatial distribution of the neurons is determinant with regard to functional maturation. The question arises as to whether the distribution is random or nonrandom. METHODS: Nitrergic cells were visualized by means of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. Stained specimens were photographed, and the borders of the myenteric plexus and the nuclei of the nitrergic neurons were digitalized. Plexus Pattern Analysis software was used to count the nuclei of nitrergic neurons, calculate the proportions of the areas covered by the plexus and the gut wall, and perform randomization analyses. RESULTS: The distribution pattern of the nitrergic neurons changed markedly between weeks 14 and 22 of gestation. The nitrergic neurons were randomly distributed at week 14 but were aggregated in the plexus and within the individual ganglia at week 19. The dynamics of these changes exhibited regional differences. CONCLUSIONS: The results suggest that, in addition to the gut wall and the plexus, other intraganglionic constituents may contribute to the aggregation of nitrergic cells and such examinations should be extended to other cell types in the future.

Computer-aided morphometric analysis of the developing concentric structure of the human fetal intestinal tube.

The Image-Pro Plus 3.0 morphometric program was used to study the region-specific organization of the human fetal intestine across the radial axis of the gut at weeks 12 and 18 of gestation. The thicknesses of the epithelium, the submucosa, the muscular layers and the myenteric ganglia were measured in resin-embedded semithin sections. Statistical analysis of the collected data was performed by using the two-way ANOVA, the SNK test and the Pearson correlation. The structural changes relating to the gut morphogenesis within this developmental period were followed both light and electron microscopically. The various tissues forming the radial axis of the intestinal tube exhibited different trends concerning their individual development. The thickness of the epithelium did not change in the fetal period investigated, although the epithelial surface displayed characteristic ultrastructural changes. The thickness of the submucosal layer increased significantly, but with different dynamics along the longitudinal axis, whereas the increases in size of the muscular layers and the myenteric ganglia did not differ significantly along the longitudinal axis of the embryonic intestine. The Pearson correlation revealed a significant correlation between the development of the circular muscle layer and that of the myenteric plexus along the whole length of the intestinal tube. The epithelium, the submucosa and the longitudinal muscle layers developed independently between weeks 12 and 18 of gestation.

Contraceptive efficacy of daily administration of 0.5 mg mifepristone.

The antiprogestin mifepristone has shown potential to be used as a contraceptive. If 200 mg mifepristone is administered immediately after ovulation, the endometrium shows sufficient impairment of secretory development to prevent implantation. Low daily doses of mifepristone have been shown to reduce several of the local factors regarded as crucial for implantation in human endometrium. To find out if this regimen is sufficient to prevent pregnancy, 32 women were recruited for a study where 0.5 mg mifepristone was administered daily. A total of 141 cycles were studied. Five pregnancies occurred, which was significantly less than if no contraceptive method had been used. However, the dose chosen did not seem sufficient to act as a contraceptive although it is probably not possible to increase the dose without disturbing ovulation and bleeding pattern.

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).

Effects of long-term low-dose mifepristone on reproductive function in women.

Low-dose antiprogestin administration has been proposed as a new contraceptive modality to interference with endometrial receptivity without disturbing ovarian function. The effects of 1 mg/day mifepristone for 150 days on the menstrual cycle were assessed in 21 surgically sterilized women. The aim was to study each woman for one control cycle and during months 1, 3 and 5 of treatment. Ovulation, endometrial thickness, serum oestradiol and progesterone, urinary luteinizing hormone, endometrial morphology and cervical mucus were assessed. Luteal phase progesterone concentrations were observed in 36 of the 60 treated months assessed and less frequently as treatment progressed. The bleeding pattern was regular in most biphasic cycles, while prolonged interbleeding intervals or no bleeding were associated with monophasic cycles. Altered endometrial morphology was found in all cases irrespective of the occurrence of luteal activity. Increased endometrial thickness and dilated glands were observed in 25 and 34% respectively of the monophasic cycles. Mifepristone, 1 mg/day, interferes with endometrial development while allowing the occurrence of biphasic ovarian cycles and regular bleeding. However, it also prevents ovarian cyclicity in a high proportion of treated months, and this is associated with increased endometrial growth in some women, which may be of concern.

PIP: Low-dose antiprogestin administration has been proposed as a new contraceptive modality that interferes with endometrial receptivity without disturbing ovarian function. To explore this potential, the effects on the menstrual cycle of 1 mg/day of mifepristone for 150 days were assessed in 21 surgically sterilized women from Santiago, Chile. Control cycles were biphasic in all 21 women and ovulatory in 20 women. Luteal phase progesterone concentrations were observed in 36 of the 60 treatment months (1, 3, and 5) assessed. The proportion of ovulatory cycles was highest during month 1 and decreased progressively with treatment. 40% of treatment cycles were monophasic and bleeding cyclicity was altered in 57%. Prolonged inter-bleeding intervals or no bleeding occurred in monophasic cycles. Endometrial morphology was altered in all cases, regardless of the occurrence of luteal activity. Increased endometrial thickness and dilated glands were recorded in 25% and 34%, respectively, of the monophasic cycles. These findings suggest that 1 mg of mifepristone interferes with endometrial development while allowing biphasic ovarian cycles and regular bleeding. Whether these endometrial alterations are sufficient to prevent implantation remains to be established. The long-term effect of prevention of ovarian cyclicity and the associated increased endometrial growth recorded in some women require further investigation.

Sequential pattern of nerve-muscle contacts in the small intestine of developing human fetus. An ultrastructural and immunohistochemical study.

The developing enteric nervous system of the human fetus has been studied by means of electron microscopy and neuron-specific enolase immunocytochemistry between the 10th and 26th week of gestation, with special reference to the development of nerve-muscle contacts. In the 10th week of gestation the circular muscle layer is formed, followed by the appearance of a primitive myenteric plexus, and the longitudinal smooth muscle layer in the 12th week of gestation. Adherens-type junctions between the smooth muscle cells and the elements of the myenteric plexus, interdigitation of nerve and muscle processes, and also contacts without any morphological specialization are frequent until the 18th week, when the mechanical points of attachment are relocated from the circular to the longitudinal muscle layer. By this time the developing myenteric plexus becomes ensheathed by non-neuronal cells, disrupting the direct contacts between smooth muscle cells and the primary strands of the myenteric plexus. The possible functional significance of these changing nerve-muscle contacts is discussed in the present paper.

Histochemical and ultrastructural features of the developing enteric nervous system of the human foetal small intestine.

The developing enteric nervous system of the human foetus has been analyzed at the 10th and 18th week of gestation with a special reference to the development of nerve-muscle contacts. The myenteric plexus formation was analyzed by means of electron microscopy and on whole-mounts after NADH diaphorase histochemistry. The development of noradrenergic innervation as an extrinsic inhibitory supply was followed by means of a glyoxylic acid-induced fluorescence method. Differentiated neurons and neuroblasts both occurred in myenteric ganglia of the 10- and 18-week-old foetus although the ganglionic neuropil was almost unidentifiable ultrastructurally at the 10th week of gestation but was mature looking at the 18th week. The nerve plexuses connecting the ganglia frequently formed distant and close myoneural contacts. NADH-diaphorase histochemistry revealed that in the 18-week-old human foetus most of the neural perikarya was within the ganglia. Also, at the 18th week of gestation a well-defined fluorescent network was demonstrated within the ganglia, as well as in the internodal segments. On the basis of these observations we concluded that the time between the 10th and 18th week of gestation has paramount importance for both the morphological and functional maturation of the enteric nervous system.

Local treatment of colpitis with Betadine vaginal suppository.

Observations made with Betadine vaginal suppository in the treatment of 60 women suffering from colpitis have been discussed. The results of microbiological examinations of vaginal discharge sampled before 1 week and 4 weeks after the onset of therapy were compared. The effectiveness, tolerance of therapy, and the subjective opinion of the treated women were analysed. According to the results of the examinations in response to Betadine vaginal suppository the positivity of vaginal discharge for Candida decreased from 16 to 3 cases, for Trichomonas from 8 to 1 case, for aerobic bacteria from 16 to 7 cases, and the occurrence of mixed infections from 20 to 2 cases. Subjective complaints, burning, stinging sensation were rapidly moderated. In 52 of the 60 women discharge ceased, in another 4 cases it significantly decreased. In 51 cases burning sensation in the vagina ceased, and in 5 women it significantly decreased. Recurrence was not observed when a control examination was done after 1 month. The women tolerated Betadine vaginal suppository well and found it effective and easily applicable. At the beginning of treatment burning sensation was aggravated in 3 cases. These patients refused to further participate in the examination and received systemic treatment. Due to its broad bactericidal, fungicidal, protozoicide action and its good tolerability Betadine suppository was found to be useful for the local treatment of colpitis.

Betaloc therapy in pregnancy-associated mild and moderately severe hypertension.

In a prospective study the effect of Betaloc therapy on pregnancy-associated mild and moderately severe hypertension has been examined. Besides the effects on the mother the changes of foetal heart rate were also observed. The antihypertensive therapy was performed with daily 3 x 100 mg (3 x 1 tab) oral doses of Betaloc in 30 pregnant women with 140-170 mmHg systolic and 90-110 mmHg diastolic blood pressure values at admittance. In the course of the examinations definite and statistically significant fall of systolic flood pressure could be observed following the onset of Betaloc therapy as early as on the first day of drug administration which could be maintained during therapy. Somewhat less definite but the same change was observed in the diastolic blood pressure. Maternal pulse rate was significantly higher before therapy than after finishing Betaloc therapy. Notable foetal bradycardia was not observed, in the course of Betaloc administration the decrease of foetal heart rate did not surpass the normal limit values. During the therapy no side-effect attributable to the drug was observed.

[Effect of caffeine on the contractility of the fetal heart muscle]. / Die Wirkung des Koffeins auf die Kontraktilität des fetalen Herzmuskels.

The caffeine-induced changes in contractility of early human fetal hearts obtained from termination of pregnancy and surviving in nutrient solution was studied. Three concentrations of caffeine (0.01; 0.1 and 1 mMol/l) were applied. In the presence of caffeine there was a significant age- and concentration-dependent increase in the contractility of fetal hearts. On the basis of this positive inotropic and the previously observed tachycardiac effect of caffeine the authors call attention to the potential harmful effect of the drug on the fetal heart.

[Prenatal effect of fenoterol (Partusisten) on the heart in the presence of verapamil]. / Pränatale Herzwirkungen des Fenoterol (Partusisten) in Gegenwart von Verapamil.

The effect of fenoterol and fenoterol + verapamil on fetal hearts obtained from termination of pregnancy and surviving in nutrient solution was investigated. In 10--20-week-old fetal hearts, under the influence of fenoterol, a concentration-dependent increase was observed in spontaneous rate and contractility. A close linear correlation was found between the fetal age and the tachycardiac effect of the drug. The positive chronotropic and inotropic effect of fenoterol was significantly decreased by a therapeutically meaningful concentration (20 micrograms/l) of verapamil. The results are in favour of the suggestion that the inhibition by verapamil of the in utero fetal tachycardiac effect of fenoterol might occur, at least in part, in the fetal heart itself.

[Effect of caffeine on fetal heart rate and caffeine consumption habits in pregnant patients]. / Die Wirkung des Koffeins auf die fetale Herzfrequenz und die Koffeinkonsum-Gewohnheiten der Schwangeren.

Responsiveness to caffeine was studied in early human fetal hearts surviving in nutrient solution. Alterations of the spontaneous contraction rate were followed. Three concentrations (0.01, 0.1 and 1 mmol/l) were applied. In the presence of caffeine there was a significant age- and concentration-dependent increase in fetal heart rate in the period of the 6th to 17th weeks of gestation. Concerning the caffeine consumption habits of pregnant women a questionaire investigation was carried out in 686 cases. 69 per cent of the pregnants regularly drink coffee; 10.7 per cent of them consumes more than 300 mg per day. The authors call attention to the potential harmful effect of caffeine on the fetal heart.

Termination of very early pregnancy by RU 486--an antiprogestational compound.

RU 486, a new antiprogestational compound, was given to 37 women seeking termination of pregnancy and with amenorrhea of 42 days or less. One patient was found at the second follow-up visit to have an extrauterine pregnancy. The patients received either 25 mg, 50 mg or 100 mg RU 486 twice daily for four days. All patients attended three follow-up visits, one, two and five to six weeks after the start of therapy. The start, duration and amount of bleeding as well as plasma progesterone, beta-hCG and cortisol concentrations were determined for each treatment day and at the follow-up visits. All patients but three started to bleed during treatment. Frequency of complete abortion was 61% (22 out of 36 patients). In only three patients was the pregnancy unaffected by treatment. The clinical efficacy of the treatment was not dose-dependent. Most of the patients experienced only minor side effects in terms of mild uterine pain, nausea and vomiting. However, two patients suffered from heavy bleeding requiring blood transfusion and curettage. In the patients with complete abortion, beta-hCG values decreased significantly but not until the first follow-up visit. The plasma progesterone also decreased. The decrease appeared earlier with the higher daily dose of RU 486. Cortisol concentrations increased during treatment with all 3 dosage regimens but the levels remained within the normal range. It is concluded that treatment with RU 486 may provide a novel therapy for "menstrual regulation" but the efficacy of the treatment needs to be improved to compete with alternatives such as vacuum aspiration.

PIP: RU 486, a new antiprogestational compound, was given to 37 women seeking termination of pregnancy and with amenorrhea of 42 days or less. 1 patient was found at the 2nd follow-up visit to have an extrauterine pregnancy. The patients received either 25 mg, 50 mg, or 100 mg RU 486 twice daily for 4 days. All patients attended 3 follow-up visits, 1, 2, and 5-6 weeks after the start of therapy. The start, duration, and amount of bleeding as well as plasma progesterone, beta-human chorionic gonadotropin (hCG) and cortisol concentrations were determined for each treatment day and at follow-up visits. All but 3 patients started to bleed during treatment. Frequency of complete abortion was 61% (22 of 36 patients). In only 3 patients was the pregnancy unaffected by treatment. The clinical efficacy of the treatment was not dose-dependent. Most of the patients experienced only minor dise effects in terms of mild uterine pain, nausea, and vomiting. However, 2 patients suffered from heavy bleeding requiring blood transfusion and curettage. In patients with complete abortion, beta-hCG values decreased significantly but not until the 1ft follow-up visit. The plasma progesterone also decreased. This decrease appeared earlier with the higher daily dose of RU 486. Cortisol concentrations increased during treatment with all 3 dosage regimens but the levels remained within the normal range. It is concluded the treatment with RU 486 may provide a novel therapy for menstrual regulation but the efficacy of it needs to be improved to compete with such alternatives as vacuum aspiration.

[Increase in weight and changes in the heart weight/body weight quotient of the embryonic heart in early pregnancy]. / Gewichtszunahme und Veränderung des Herzgewicht/Körpergewichts-Quotienten des embryonalen Herzens in der Frühschwangerschaft.

On the basis of measuring heart weight and body weight of 73 embryos it is shown that there is a close linear correlation between growth of the heart, increase of heart weight and body weight and age of pregnancy. The increase in heart weight, however, is not proportional to that of the increase in body weight during intrauterine development. There is a considerable shift in the heart weight/body weight ratio (i.e. a change in the cardio-fetal index: (formula; see text) In early pregnancy (5-7th weeks) the cardio-fetal index varies from 1.5 to 2.5 and in the course of pregnancy it decreases gradually. In the 12th-14th weeks it falls below 1, and following the 14th week it stays on a relatively constant level of 0.5-0.8. In early embryonic phase heart weight in relation to body weight is 2-5 times bigger than in the period following the 14th week. The significance of these findings is discussed.

[The effect of temperature changes on the spontaneous heart rate of the isolated surviving human embryonal heart]. / Zur Temperaturabhängigkeit der Spontanfrequenz des isolierten überlebenden menschlichen embryonalen Herzens.

The effect of changes in temperature on the spontaneous fetal heart rate (FHR) of 17 isolated human hearts obtained from early pregnancy was investigated. A slow increase in the temperature of the nutrient solution provoked a 10/min FHR increase pro every 1 degrees C change from 32 to 38 degrees C, and this change did not depend on weight and age of the hearts and the basic FHR. At 40 degrees C the tendency of FHR decrease could be observed especially in case of bigger hearts. As result of the decrease in the temperature of the nutrient solution the fetal hearts stopped beating between 22 and 12 degrees C.

[Effect of adrenaline, noradrenaline, isoproterenol and tyramine on the isolated surviving human fetal heart]. / Die Wirkung von Adrenalin, Noradrenalin, Isoproterenol und Tyramin am isolierten überlebenden Herzen humaner Feten.

Studies were conducted by the authors into the action of adrenergic stimulants on isolated surviving hearts of 64 foetuses obtained from termination of pregnancies. They found that under the direct impact of stimulating catecholamines (adrenaline, noradrenalinee, and isoproterenol) the adrenergic receptors caused increase in spontaneous frequency of heart contractions, as early as in the fifth to ninth weeks of development, that is before adrenergic innervation of the heart. Such tachycardic effects were increasingly pronounced between the fifth and 24th week, along with growing age. Reactions produced by early and somewhat developed hearts were different with significance. Convincing effects were not detectable until hearts were ten or eleven weeks of developmental age, when tyramine was used which acted through release of noradrenalin. The latter date was in approximate coincidence with the appearance of adrenergic nerve elements for growth into the cardiac substance. In the 17th week of development, tyramine was used with good success to produce numerous effects which were characteristic of catecholamines, such as higher irritability and contractility of the myocardium and decrease of the effective refractory phase.

[Method for determination of mechanical activity and electrophysiological properties of isolated human embryonal heart]. / Verfahren zur Bestimmung der mechanischen Aktivität und der elektrophysiologischen Eigenschaften isolierter menschlicher embryonaler Herzen.

The authors have invented a complex method by which to examine surviving hearts of early human embryos. The method enables testing of mechanical activity of the heart as well as of many electrophysiological parameters, such as spontaneous heart rate, electrical threshold of stimuli, stimulus conduction time, effective refractory phase, and intracellular action potential. --A special transport device was designed for storage and movement of embryonic hearts which were all obtained from cases of legal abortion. A contractility recorder was devised to follow up mechanical heart activity. A variant of the standard micro-electrode technique was developed to measure electrophysiological parameters. This technique has worked extremely well in recording the membrane potential at rest and the intracellular action potential in cells of small embryonic hearts. Also developed was a differentiation unit for direct and convenient measurement of depolarisation velocity of heart cells. The complex method has proved to be particularly suitable to study physiological characteristics relating to the responsiveness of the early human foetal heart to pharmaceuticals.

[Action of acetylcholine and carbamylcholine on spontaneous rates of hearts isolated from human embryos]. / Die Wirkung von Azetylcholin und Karbamylcholin auf die Spontanfrequenz des isolierten menschlichen embryonalen Herzens.

Fifty-eight early human foetal hearts, which had been obtained from cases of legal termination of pregnancy, were studied for their sensitivity to acetylcholine. Acetylcholine action was found to cause moderate decline in spontaneous human embryonic heart rate around the fifth and sixth weeks of development. The sensitivity to acetylcholine of the embryonic heart, aged between five and eight weeks, was found to go up along with growing age. The action of carbamylcholine, another cholinergic stimulant, was studied in some of the cases. Bradycardic action of carbamylcholine upon hearts aged between nine and ten weeks was found to be much stronger than that on hearts aged between five and six weeks. --The findings recorded from these studies have produced evidence to the effect that parasympathetic innervation in human embryonic hearts was preceded by the appearance of cholinergic receptors. They have also shown that in a very early phase of ontogenesis considerable change is undergone also by the cholinergic receptors of the heart.

Induction of preterm labor in the rat by antiprogesterone.

The validity of a new concept, predicting that preterm labor can be induced without oxytocic stimulation by a regulatory imbalance, generated by antiprogesterone (A-P)-provoked P withdrawal (Pw), has been examined in the rat model. At day 19 of pregnancy a single oral dose of the steroidal A-O isoxazol, which inhibits P synthesis, significantly reduced uterine P levels, increased uterine estradiol and prostaglandin F levels, and induced preterm labor. This precocious regulatory imbalance and preterm labor were prevented, by blocking the A-P-induced Pw with P treatment. The regulatory imbalance which triggered the onset of spontaneous labor in the control animals was similar, but it occurred at term rather than before term. The potential of this method in improving the management of medically indicated induction of preterm labor is discussed.