Ventricular blood pressure and cardiac output changes in epinephrine- and metoprolol-treated chick embryos

The effects of a teratogenic dose (5 micrograms) of epinephrine on mean ventricular blood pressure (MVBP) and cardiac output (CO) at one and two hours after treating stage 24 chick embryos were investigated. Previous work demonstrated that a differential response in terms of cardiac rhythm during the first hour after epinephrine treatment was related to pathogenesis of two contrasting types of aortic arch malformations. Absence of one or more aortic arches occurred more frequently in embryos which developed a characteristic dysrhythmia, while persistence of the left fourth aortic arch (PL4AA) occurred more frequently in nondysrhythmic embryos. In this study, dysrhythmic epinephrine-treated embryos exhibited reductions in both MVBP and CO at one hour after treatment when compared to control values. Nondysrhythmic epinephrine-treated embryos exhibited elevated MVBP and no change in CO at one hour after treatment. MVBP and CO in recovered dysrhythmic and nondysrhythmic embryos were similar to control values at two hours following epinephrine treatment. MVBP and CO measurements were obtained from embryos which were pretreated with metoprolol and then subsequently treated with epinephrine. Metoprolol is a beta 1-adrenoreceptor antagonist which was previously shown to block the teratogenic effects of epinephrine and other catecholamines with beta 1-adrenoreceptor agonist properties. Pretreating embryos with metoprolol in this study reduced the dysrhythmogenic potential of epinephrine and also blocked the MVBP and CO changes observed in embryos treated with epinephrine alone. We conclude that pathogenesis of 1) abnormally absent aortic arches is related to dysrhythmogenesis, reduced MVBP, and reduced CO, and 2) an abnormally persistent left fourth aortic arch is related to elevated MVBP in the epinephrine model.     

Tissue and plasma levels of a teratogenic dose of dopamine in the chick embryo following pretreatment with metoprolol or phosphate buffered saline

Metoprolol pretreatment has been shown to reduce the cardiovascular malformation rate produced by topical doses of dopamine in the stage 24 chick embryo. We report on the tissue and plasma levels and teratogenic effect of dopamine hydrochloride following topical application of a teratogenic dose in stage 24 chick embryos pretreated with either metoprolol tartrate or phosphate buffered saline (PBS). Pretreatment with either metoprolol or PBS resulted in similar patterns of dopamine distribution in the head, body, and heart, with peak levels occurring at 12 hours after dopamine treatment. Plasma concentrations of dopamine were similar for both PBS and metoprolol pretreated embryos, with plasma levels exceeding tissue concentrations, but also peaking at 12 hours after dopamine treatment. Pretreatment with PBS followed by a teratogenic dose of dopamine resulted in a decrease in the teratogenic effect of dopamine similar to that found in previous work in our lab with pretreatment with metoprolol. The developing chick cardiovascular system experiences peak susceptibility to the teratogenic effects of dopamine at stage 24 during development, which represents a time frame of about 12 hours. A delay in the peak levels of dopamine to 12 hours after dopamine treatment as compared to previous work in our lab reporting peak levels of dopamine at 1 hour, suggests that the previously reported antiteratogenic effects of metoprolol may be due, at least in part, to a delayed absorption of dopamine past the time of peak susceptibility of the embryo to the teratogen.     

Lignocaine Therapy after Acute Myocardial Infarction

Thirty-five patients with ventricular dysrhythmias and seven with other dysrhythmias after acute myocardial infarction were treated with intravenous lignocaine.Satisfactory initial suppression of ventricular ectopic beats was achieved in 27 (82%) of 33 patients after either a 50-mg. bolus or a 50-mg. bolus followed by a 100-mg. bolus of intravenous 2% lignocaine. Continuous suppression of ventricular ectopic beats was accomplished in 21 (78%) of these 27 patients by continuous intravenous lignocaine infusions of 1 to 2 mg. per minute. Recurrence of ventricular ectopic beats occurred in four patients despite lignocaine infusion rates of up to 6 mg. per minute. Six patients with ventricular ectopic beats developed ventricular fibrillation despite satisfactory initial suppression of their dysrhythmia by lignocaine. In three of them ventricular fibrillation supervened while they were receiving a lignocaine infusion and two subsequently died. Unheralded ventricular fibrillation occurred in three other patients between four and seven days after completing the full course of lignocaine therapy.Toxic effects of lignocaine were minimal in patients receiving 1 to 2 mg. per minute.     

Toxic and teratologic effects of verapamil on early chick embryos: evidence for the involvement of calcium in neural tube closure

Toxic and teratologic effects of verapamil, a calcium antagonist, on chick embryos explanted at stage 8 (four-somite stage) and cultured for 6-8 hours were investigated. In general, embryos responded to verapamil in a dose-related manner. Concentrations lower than 2 micrograms/ml had no apparent effect on the development of embryos. A concentration of 15 micrograms/ml significantly increased the incidence of embryos (approximately 80% of viable embryos) with neural tube closure defects and less numerous somites. Higher concentrations (e.g., 30 micrograms/ml) were embryotoxic and over 90% of the embryos were either severely malformed or dead after 8 hours of incubation. Compared to controls, verapamil-treated neuroepithelial cells had smoother apical surfaces and less conspicuous microfilament bundles. The deleterious effects of verapamil (15 micrograms/ml) could be reversed by subculturing the affected embryos, within 3 hours of treatment, on nutrient medium alone or on nutrient medium containing 25 micrograms/ml chlorotetracycline (CTC), a calcium agonist, the latter being more effective provided that treatment did not exceed 4 hours. Exposure of the developing neuroepithelium to 15 micrograms/ml verapamil for 3-4 hours resulted in a significant reduction in free Ca2+ levels, as revealed by the pyroantimonate precipitation method, throughout neuroepithelial cells. Overall results suggest that verapamil causes neural tube closure defects by reducing intracellular free Ca2+ levels, thereby relaxing apical microfilament bundles of developing neuroepithelial cells.     

Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4?µg/mL demecolcine, 2?mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.     

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.     

Ambulatory blood pressure during once-daily randomised double-blind administration of atenolol,metoprolol, pindolol,and slow-release propranolol

Intra-arterial ambulatory blood pressure was measured over 24 hours, in 34 patients with newly diagnosed hypertension, both before and after double-blind randomisation to treatment with atenolol (n=9), metoprolol (n=9), pindolol (n=9), or propranolol in its slow-release form (n=7). The dosage of each drug was adjusted at monthly clinic visits until satisfactory control of blood pressure was achieved (140/90 mm Hg or less by cuff) or the maximum dose in the study protocol was reached. A second intra-arterial recording was made after these drugs had been taken once daily at 0800 for three to eight months (mean 5·0±SD 1·4) and was started four hours after the last dose.At the end of the 24-hour recordings blood pressure was significantly lower with all four drugs. The extent to which the drugs reduced blood pressure, however, differed over the 24 hours. Atenolol lowered mean arterial pressure significantly throughout all 24 recorded hours, metoprolol for 12 hours, pindolol for 15 hours, and slow-release propranolol for 22 hours. Neither metoprolol nor pindolol lowered blood pressure during sleep. A significant reduction in heart rate was observed over 20 hours with atenolol, 20 hours with metoprolol, 10 hours with pindolol, and 24 hours with slow-release propranolol. Atenolol, metoprolol, and slow-release propranolol continued to slow the heart rate 24 hours after the last tablet was taken; this effect on heart rate, however, was not sustained throughout the second morning in those patients taking atenolol. Pindolol, the only drug studied that has intrinsic sympathomimetic activity, increased heart rate and did not lower blood pressure during sleep.Atenolol and slow-release propranolol are effective as antihypertensive agents over 24 hours when taken once daily, whereas metoprolol and pindolol may need to be taken more frequently. At times of low sympathetic tone, however, such as during sleep, beta-blockers with intrinsic sympathomimetic activity may raise heart rate and attenuate the fall in blood pressure with treatment.     

Amphetamines reduce embryonic size and produce caudal hematomas during early chick morphogenesis

Experiments were designed to study some of the similarities and differences in the effects of amphetamines and trypan blue on early chick morphogenesis. Both dextroamphetamine sulfate (0.5 mg/egg) and methamphetamine hydrochloride (1.0 mg/egg) were capable of inducing, in 3-day chick embryos, caudal hematomas which were similar in appearance and location to those routinely observed following treatment with trypan blue. It was found, too, that both dextroamphetamine and methamphetamine treated embryos frequently exhibited a significant decrease in crown rump length and cross-sectional area of the notochord, neural tube, dorsal aortae and whole body section, when compared with unopened or saline injected controls. Trypan blue treated embryos had only a rare decrease or increase in the size of structures when compared to either control group. These findings suggest that the amphetamines have an ability to decrease or retard embryonic growth in the chick.     

Postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned pig embryos by increasing nuclear retention and formation of single pronucleus

The objective of this study was to investigate the effects of postactivation treatment with nocodazole on morphologic changes of donor nuclei and in vitro and in vivo development of somatic cell nucleus transfer (SCNT) embryos in pigs (Sus scrofa). Somatic cell nucleus transfer oocytes were either untreated (control) or treated with nocodazole or demecolcine after electric activation, then cultured in vitro or transferred to surrogate pigs. Treatment with nocodazole (30%) and demecolcine (29%) after electric activation improved embryo development to the blastocyst stage compared with the control (16%). The rate of oocytes that formed single clusters of chromosomes or a pronucleus 4 h after activation was higher after treatment with nocodazole (82%) and demecolcine (86%) than under the control conditions (66%), and this tendency was not altered even 12 h after activation. Pseudo-polar body extrusion was inhibited by nocodazole and demecolcine, and the rate of embryos with diploid chromosomes was higher after treatment with nocodazole (86%) and demecolcine (77%) than under control conditions (58%). Nocodazole treatment resulted in a farrowing rate of 50% with a 1.7% efficiency of piglet production, whereas controls showed a farrowing rate of 60% and a production efficiency of 3.8%. Our results demonstrate that postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned embryos likely by increasing nuclear retention and formation of single pronuclei. In vivo development could be achieved from the transfer of nocodazole-treated embryos but showed some defects compared with control.     

Pathogenesis of persistent truncus arteriosus induced by nimustine hydrochloride in chick embryos

Nimustine hydrochloride (ACNU) is a nitrosourea derivative anticancer agent which has been shown to cause persistent truncus arteriosus in chick embryos. The objective of this study was to confirm the teratogenic effects of ACNU on the cardiovascular system of chick embryos and to determine whether ACNU induces persistent truncus arteriosus by interfering with neural crest cells. Various doses of ACNU ranging from 10 to 200 micrograms were injected under the chorioallantoic membrane of chick embryos on the third day of incubation. Saline solution was used as the control. After 10 to 11 days of incubation, 242 (46%) survivors of the 524 treated eggs were obtained. The survival rates of the embryos and the frequencies of cardiovascular anomalies were dose dependent. Of 146 embryos with cardiovascular anomalies, 104 (71%) had persistent truncus arteriosus. Ventricular septal defect and double-outlet right ventricle were seen in 37 (25%) and one (1%), respectively. Aortic arch anomalies were seen in 116 embryos (79%). Quail-chick chimeras (chick embryos with quail cardiac neural crest) were treated with 50 micrograms of ACNU and examined histologically 24 hours later. These chimeras showed dying neural crest cells in the pharyngeal arches. Dying cells were also noted in the neural tube, cranial ganglia, retina, and otocyst. These results suggest that persistent truncus arteriosus in chick embryos treated with ACNU is induced by neural crest cell death.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Effect of cyclosporin A on rat kidney catecholamines

The immunosuppressive agent, Cyclosporin A, (CsA) has been associated with nephrotoxicity and hypertension. The mechanism for these effects are not known. We therefore determined the levels of the catecholamines; epinephrine (EPI), norepinephrine (NE) and dopamine (DA) and some of their metabolites; epinine, dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), metanephrine (ME) and 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the kidneys of rats treated intraperitoneally with either CsA (120 micrograms/kg/body wt/day) or control vehicle (1 ml olive oil/kg body wt/day). Six control or CsA treated rats were sacrificed at 1 hour or 24 hours after a single treatment or after 7 days of daily treatment. Renal catecholamine levels were determined using HPLC-amperometric detector. Treatment with CsA increased renal NE and EPI levels by 59% and 70% respectively within 1 hour. In the rats sacrificed 24 hours after treatment, renal NE, EPI and DA levels were similar to or less than the control levels. Treatment with CsA for 7 days resulted in marginal increases in renal NE (22%) and EPI (30%). These changes were associated with a significant decrease in the levels of catecholamine metabolites in the CsA treated kidneys as compared to the controls. The above findings suggest that increases in renal catecholamines may be involved in the CsA-induced hypertension and nephrotoxicity, perhaps by increasing renovascular resistance.     

The effects of minoxidil on the development of the chick embryo

The effects of minoxidil were studied on chick embryos of 24 and 48 hours of incubation. Minoxidil (3%) was injected into the air sacs of the eggs at doses of 20, 30, 40, and 50 microliters per egg. The controls received 100 microliters of physiological saline. All the embryos, including controls, were examined at day 13. The total number of eggs used in this study was 300. At 24 hours incubation, the percentage of survival ranged from 87 to 21 as the dosages of minoxidil were increased from 20 microliters to 50 microliters per egg (controls = 87%). The survival of the embryos ranged from 79% to 9% after the 48-hour treatment with the similar dosages of minoxidil utilized for the 24-hour group (controls = 83%). A low incidence of gross malformations such as twisted limbs, abnormal beak, short neck and everted viscera were observed; however, the increased incidence was not statistically significant when compared to controls. Body hemorrhage and edema were of high occurrence among the treated embryos. These effects are probably secondary to the known pharmacological effects of minoxidil. The frequency and types of gross malformations did not vary much in the 24 or 48-hour treated groups.     

Effects of corticosterone on brain cholinergic enzymes in chick embryos

The effects of corticosterone on the cholinergic enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the chick embryonic brain. Chick embryos received either 0.25, 0.5, or 1.0 g of corticosterone via the air sac daily for three days during either embryonic days 6 through 8 (E6-E8), of cerebral neurogenesis, or days 10 through 12 (E10-E12), a period of cerebellar neurogenesis. Enzyme activities were determined in cerebral hemispheres, optic lobes, cerebellum and remaining brain at 10, 15, and 20 days of incubation. In embryos treated from E6 to E8, ChAT activity was generally higher at day 10 in cerebral hemispheres and optic lobes (cerebellum was not determined) while AChE activity was not affected. At day 20 ChAT activity of treated chick embryos was lower in the cerebral hemispheres and optic lobes, but not in the cerebellum; AChE activity was higher in the cerebral hemispheres, lower in the optic lobes, and not changed in the cerebellum as compared to controls. However, in embryos treated from E10 to E12 both cerebellar ChAT and AChE activities were higher at day 15 in comparison to controls. These data show that the hormonal effects were most prominent only in the brain areas undergoing neurogenesis during the period of hormonal treatment. Since AChE activity is also present in nonneuronal cells, the observed alterations caused by corticosterone may reflect glial cell responses to the hormone. Whether the hormone affects the final number and/or maturation of cholinergic neurons and/or glial cells remain to be investigated.     

Post‐activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei

The objective of this study was to examine the effect of cytochalasin B (CB) and/or demecolcine (Dc) on the remodeling of donor nuclei, nuclear ploidy, and development of somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) pig embryos. SCNT and PA oocytes were either untreated (control), or treated with CB, Dc, or both CB and Dc after electric activation, and then cultured or transferred to surrogates. In SCNT, blastocyst formation was higher after treatment with CB and/or Dc (26–28%) than in the controls (16%). The number of oocytes that formed a single pronucleus (PN) was higher after treatment with Dc (86%) and CB + Dc (86%) than under control conditions (44%) or after treatment with CB (63%). In PA, blastocyst formation was higher after CB treatment (47%) than under control conditions (28%), while the formation of a single PN was higher after treatment with Dc (88%) and CB + Dc (84%) compared to controls (34%). The rate of formation of diploid embryos was higher after treatment with Dc and CB + Dc than under control conditions. Dc treatment resulted in a farrowing rate of 50% with 1.1% production efficiency, while controls showed a farrowing rate of 37.5% and a production efficiency of 0.7%. The results of our study demonstrate that post‐activation treatment with Dc improves preimplantation development and supports normal in vivo development of SCNT pig embryos, probably because Dc induces formation of a single PN and this leads to normal nuclear ploidy. Mol. Reprod. Dev. 76: 611–619, 2009. © 2008 Wiley‐Liss, Inc.     

The effects of 5-bromodeoxyuridine (BrdU) on morphogenesis of the early chick embryo

Summary The effects of BrdU (3×10^–4 M) on morphogenesis of the chick embryo explanted at the definitive streak stage and cultured for 24 hours were studied. Compared to controls treated embryos often showed (1) an open neural tube and (2) less numerous somites. Heart development was not significantly affected by BrdU. The damage caused by BrdU was not permanent, i.e., the embryos retained the ability to undergo fairly normal morphogenesis when, after 4–5 hours of BrdU treatment, they were subcultured on a medium with excess thymidine.This study was supported by a grant from the Rutgers University Research Council No. 07-2189.     

Response of chick cerebellum in culture to L-dopa and estradiol

Cerebellar explants were removed from chick embryos at either 14 or 20 days and maintained in organ culture for 2 or 4 hours in the presence of either L-dopa or estradiol dipropionate or a combination of both. The activities of AChE and BuChE were higher in explants cultured in L-dopa, but not changed in explants cultured in estradiol. These increases were no longer seen with simultaneous treatment with L-dopa and estradiol. Moreover, simultaneous treatment decreased BuChE activity in explants from 14 day old embryos. These data suggest that estradiol may interfere with the action of neurohumors on glial cells.     

Effect on intra-arterial blood pressure of slow release metoprolol combined with placebo or chlorthalidone.

Thirty patients with essential hypertension participated in a double blind crossover trial in which they were randomly allocated to treatment with either once daily slow release metoprolol (200 mg) with placebo or once daily slow release metoprolol (200 mg) with chlorthalidone (25 mg). Ambulatory intra-arterial blood pressure was recorded continuously for 24-48 hours before treatment and two months after each change in regimen. The response of blood pressure and pulse rate to a standard exercise protocol that included supine rest and tilt, isometric, and dynamic bicycle exercise was measured during the same recording periods. Both treatments appreciably reduced blood pressure and pulse rate; mean daytime intra-arterial blood pressure was reduced from 174/95 mm Hg to 158/85 mm Hg by metoprolol plus placebo and to 143/78 mm Hg by metoprolol plus chlorthalidone. This reduction with the combined treatment was significantly greater than with metoprolol and placebo (p systolic = 0.001, p diastolic = 0.004). Mean night time pressures were reduced from 148/78 mm Hg to 139/75 mm Hg by metoprolol plus placebo and to 116/61 mm Hg by metoprolol plus chlorthalidone. Again the reduction in blood pressure was significantly greater with combined treatment (p less than 0.001) than with metoprolol plus placebo. Once daily slow release metoprolol is effective in controlling blood pressure, but this effect is greatly enhanced by the addition of a diuretic.     

Changes in the mouse neuroepithelium associated with cadmium-induced neural tube defects

The aim of this study was to investigate the teratogenic action of cadmium (Cd) on the developing mouse CNS. Pregnant mice were injected with 4 mg/kg CdCl2 on day 7, 8, 9, or 10 of gestation. These animals and saline injected controls were sacrificed either on the day before birth or at various times up to 48 hours after injection and the embryos examined grossly and histologically. Exencephaly occurred after Cd treatment on day 7 or 8 and its development was examined in day 8 embryos. Eight hours after Cd injection many cells of the closing neural plate contained dense-staining inclusions, thought to be autophagic vacuoles. After 24 hours this damage had almost disappeared, but the anterior neural folds, although looking histologically normal, were more open than in controls. Forty-eight hours after injection it was apparent that this part of the neural tube was not going to close and would result in exencephaly. Cd exposure on day 9 or 10 did not cause gross CNS defects such as exencephaly. On both days, twelve hours after Cd injection, similar dark-staining inclusions were seen in many cells throughout the CNS. After twenty-four hours there were variable amounts of cell death, resulting in some embryos in breakdown of parts of the wall of the neural tube. Forty-eight hours after treatment all inclusions and cellular debris had disappeared, indicating repair had taken place, but in some embryos, treated on day 9, severe lasting damage was seen as dorsal openings in the previously closed neural tube.