Absorption spectra of corneas in the far ultraviolet region.

PURPOSE: To study the corneal absorption in the far ultraviolet (UV) region between 260 and 190 nm. METHODS: Thirty-four corneal samples of thickness near 20 microns were obtained from 18 porcine corneas and six human corneas with a microtome-cryostat. The authors conducted absorbance measurements of the sectioned corneal samples supported by two UV optical windows from 350 to 190 nm using a dual-beam spectrophotometer. Three whole porcine corneas were used to study the effect of freezing on the absorbance from 350 to near 290 nm. RESULTS: The absorption spectra of porcine and human corneas from 350 to 190 nm were measured and three segments in the spectrum between 260 and 190 nm have been identified. The linear absorption coefficients were determined to be 2300 +/- 330 (cm-1) at 210 nm and 2410 +/- 370 (cm-1) at 193 nm for the porcine corneas and 2320 +/- 470 (cm-1) at 210 nm and 2340 +/- 150 (cm-1) at 193 nm for the human corneas. CONCLUSIONS: A "window of ablation" in the far UV region between 220 and 190 nm has been identified in which short laser pulses of similar durations and different wavelengths may be interchangeable to ablate the corneal surface with similar characteristics.

Optical properties of porcine skin dermis between 900 nm and 1500 nm.

The weak absorption of shortwave infrared light by skin tissues between 700 and 1500 nm offers an important window for diagnosis by optical means. The strong scattering of shortwave infrared light by the skin, however, presents a challenge to the modelling of light propagation through the skin and the understanding of skin optics. We have measured the collimated and diffuse transmittance and diffuse reflectance of porcine skin dermis samples within 30 h post-mortem. Monte Carlo simulations have been performed to inversely determine the absorption coefficient, scattering coefficient and anisotropy factor of the dermis samples in the spectral range from 900 to 1500 nm. We further analyse the sensitivity of the values of the parameters to the experimental errors and inverse calculation procedures. The state of the cellular integrity of the skin samples following optical measurements was verified using transmission electron microscopy. These results were correlated to study post-mortem effects on the in vitro optical properties of porcine dermis. We concluded that for samples stored within crushed ice for up to 30 h post-mortem the wavelength dependence of optical properties of the dermis remains unchanged while the values of the parameters vary moderately due to modification of the water content of the tissue.

Alcohol intake in high alcohol drinking (HAD) rats is suppressed by FG5865, a novel 5-HT1A agonist/5-HT2 antagonist.

Both the 5-HT2 antagonist, FG5606 (amperozide), and the mixed 5-HT1 agonist/5-HT2 antagonist, FG5893, attenuate significantly the volitional intake of alcohol in the cyanamide treated rat. The purpose of the present study was to investigate the effect on alcohol drinking in the selectively bred, high alcohol drinking (HAD) rat of a new and novel 5-HT1A agonist/5-HT2 antagonist, FG5865 (2-[4-[4,4-bis(4-fluorophenyl)butyl]-1-piperazinyl]-3-pyridinecarboxy lic acid methyl ester), which shares pharmacological properties with FG5893. Initially, a standard three bottle preference test for water vs. 3% to 30% alcohol solutions was given over 11 days to determine the maximally preferred concentration for each animal. Then water and this solution, which ranged between 9% and 20% with an overall mean absolute intake of 6.3 +/- 0.5 g/kg per day, was offered over three consecutive 4-day test sequences: (1) predrug control; (2) SC injections b.i.d. of either 1.0 mg/kg or 2.5 mg/kg FG5865 or saline control vehicle; and (3) postdrug. Whereas saline failed to alter alcohol consumption of the HAD rats, FG5865 caused a significant dose dependent reduction by as much as 75% in the intakes of alcohol during its administration in terms of both g/kg (p < 0.01) and proportion of alcohol to total fluid intake (p < 0.01). During the administration of 2.5 mg/kg FG5865, alcohol drinking declined from 6.5 +/- 0.3 g/kg to as low as 2.3 +/- 0.2 g/kg per day. Neither the body weight of the HAD animals nor their intake of food was affected by either dose of FG5865. These results uphold the concept that the 5-HT1A and 5-HT2 receptor subtypes in the brain play a part in the aberrant drinking of alcohol of the HAD rat. Because FG5865 influences the activity of serotonergic neurons in the mesolimbic system of the rat, it is envisaged that the drug suppresses alcohol drinking by way of its action on these neurons.

Ethanol drinking following 6-OHDA lesions of nucleus accumbens and tuberculum olfactorium of the rat.

Previous studies have shown that lesions of the dopaminergic system in the brain produced by an intracerebroventricular injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), evoke significant changes in ethanol drinking. In the present experiments, dopaminergic systems of Sprague-Dawley rats were lesioned by 6-OHDA infused into either the tuberculum olfactorium or nucleus accumbens, two of the structures implicated in drug-related reinforcement. Prior to the lesion and immediately thereafter, tests for ethanol preference were undertaken in which water was offered in a self-selection situation together with ethanol which was increased in concentration from 3-30% over a 10-day interval. Following the circumscribed ablation of dopaminergic neurons within either the N. accumbens or tuberculum olfactorium, preference for ethanol increased significantly with absolute intakes exceeding 4.0 g/kg at the 7% concentration during the first postlesion drinking test. During the second postlesion preference test, the mean consumption of ethanol exceeded 6.0 g/kg at the 11% concentration and 4.0 to 5.0 g/kg at the 20 and 30 percent concentrations offered to the rats. When adjacent areas just dorsal or lateral to these structures were lesioned by 6-OHDA, no significant change in consumption of ethanol occurred. Thus, it is envisaged that one of the functional roles for the dopaminergic neurons of the N. accumbens and tuberculum olfactorium is to regulate the craving for a drug with addictive liability such as ethanol. As a result of an impairment of normal function of dopamine receptors or a perturbation in the release of this catecholaminergic neurotransmitter, ethanol becomes reinforcing upon repeated exposure. Thus, an addictive-like state consequently ensues. Finally, it is envisaged that the control mechanism underlying the function of the dopaminergic neurons in the medial-basal forebrain is functionally disinhibited in individuals that consume ethanol to the point of abuse.

Alcohol drinking in rats is attenuated by the mixed 5-HT1 agonist/5-HT2 antagonist FG 5893.

Over the last three decades, the neurotransmitter serotonin (5-HT) has been implicated in the etiological mechanisms underlying the excessive drinking of ethyl alcohol. Recently, the 5-HT2 antagonist amperozide was found to reduce selectively the high intake of alcohol in the cyanamide-induced drinking rat without any adverse side effects. The purpose of the present study was to determine the action on alcohol drinking of the novel second-generation amperozide-like drug, which is a mixed 5-HT1 agonist/5-HT2 antagonist, FG 5893 (2-[4-[4,4-bis(4-fluorophenyl)butyl]-1-piperazinyl]-3-pyridinecarb oxylic acid methyl ester). To induce preference for alcohol in Sprague-Dawley rats, the enzyme aldehyde dehydrogenase was inhibited by cyanamide administered in the absence of alcohol in a dose of 10 mg/kg twice a day over three days. A standard three-bottle preference test was used in which water and a maximally preferred concentration of alcohol were offered to each animal. Following control tests of alcohol preference for 3 days, either a saline control vehicle or FG 5893 in a dose of 0.5, 1.0, or 2.5 mg/kg was administered subcutaneously at 1600 and 2200 for 3 consecutive days. Whereas control injections of saline were without effect on alcohol consumption, all doses of FG 5893 significantly reduced the 24-h intake of alcohol in terms of both absolute g/kg and proportion of alcohol to total fluid intake. Further, the 1.0 and 2.5 mg/kg doses of FG 5893 continued to suppress alcohol consumption over two 4-day tests immediately following the injection sequence and after a 40-day interval.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on dispersed unincubated chick blastoderm cells. II. Formation of contacts and cell sorting in aggregates of unincubated chick blastoderm and heart cells.

The configuration of blastoderm-heart cell aggregates after cell sorting depended largely on the proportion of each cell type initially present in the culture. In heterotypic aggregates, blastoderm cells were always found to partially or completely surround heart cells with both cell types retaining their characteristic morphology. Specialized junctions (e.g. desmosomes) developed only between cells of the same type. These observations suggest that blastoderm cells are unable to recognize and form stable contacts with heart cells.

Studies on dispersed unincubated chick blastoderm cells. I. Early phases of aggregation.

Early phases of aggregation of dispersed unincubated chick blastoderm cells were investigated. Dispersed cells, when maintained in appropriate cultures, began adhering to one another almost immediately and eventually formed spherical aggregates without any recognizable structures except for occasional blood islands, after 24 hr of incubation. The number and size of aggregates derived from dispersed cells depended largely upon the speed of rotation and initial cell density. Treatment with colchicine (4 microgram/ml) had no apparent effect on adhesion of cells, although it yielded smaller aggregates than controls, especially after 14 hr of incubation. Direct observation on mitotic activity, estimations of cell number and nucleic acid content, and pulse labeling experiments with 3H thymidine clearly showed that aggregates were derived entirely from adhesion of free cells and/or fusion of smaller aggregates during the first 12 to 13 hr of incubation. In addition, all cell types present in the unincubated blastoderm contributed to the formation of aggregates, not one particular cell type.

Effects of ethanol and acetaldehyde on cultured pre-implantation mouse embryos.

Pre-implantation 2-cell stage mouse embryos, obtained from superovulated CF-1 mice, were exposed to ethanol and acetaldehyde through the culture medium for 60 min followed by a 105-h incubation period. Control and ethanol exposed embryos survived equally well in ethanol concentrations as high as 800 mg/100 ml medium and acetaldehyde levels up to 10 mg/100 ml medium.

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells.

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE2 affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE2 (2 x 10(-5)M) had no effect, but as the concentration increased (3 x 10(-5)M and 5 x 10(-5)M), PGE2 did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.

Studies on cell differentiation: inducing capacity of sulfhydryl-containing amino acids on post-nodal pieces of chick blastoderms.

The inducing capacity of sulfhydryl (SH)-containing amino acids (cysteine and glutathione) on post-nodal pieces (PNPs) of stage 4 chick blastoderms was investigated. PNPs were treated for different lengths of time with chick Ringer's solution (control group) or chick Ringer's solution containing cysteine or glutathione, followed by culturing for 2-10 days on Spratt-Haas agar medium or on the chorioallantoic membrane of 8-day chick embryos. Control PNPs rarely showed differentiation, but those treated with the amino acids for six hours or longer developed structures such as neural tissue, notochord, somite mesoderm, and nephric tubules. The pulsatile tissue was only seen in the PNPs cultured for four days or longer. Two-four hours of treatment was too short to provoke induction in a statistically significant number of PNPs. The highest frequency of induction was noted in those pretreated with glutathione (8 mug/ml) for eight hours, followed by culturing for four days. The magnitude of the inducing capacity and toxicity of the amino acids were concentration dependent: a deleterious effect was observed at 14 mug/ml; the highest frequency of induction occurred at 8 mug/ml, but the frequency decreased as the concentration decreased; at 2 mug/ml all PNPs remained viable, but only a few (9-14%) showed differentiation. The inducing capacity of the amino acids was counteracted by equimolar concentrations of rho-chloromercuribenzoic acid or omega-chloroacetophenone. The effects of glutathione (8 mug/ml) differed from those of Hensen's node grafts in that the former caused sublethal cytolysis and inhibited H-3-uridine uptake in competent ectodermal cells during the first 18 hours of cultivation.

Effects of cytochalasin B on the morphogenesis of explanted early chick embryos.

Effects of cytochalasin B (CB) on chick embryos explanted at stages 4-5 and cultured for 24 hours were studied. CB (2-4 mug/ml) inhibited blastodermal expansion and neural tube closure in over 90% of the embryos. Somite formation was inhibited only where neural tissue was very degenerate. The development of other structures was usually unaffected. Cellular degeneration occurred in severely affected neuroepithelium, but cells at various phases of mitosis were found throughout its thickness, suggesting that interkinetic nuclear migration had been inhibited. Electron microscopic studies of the flattened neural tube showed that CB disrupted microfilaments and reduced the number of cytoplasmic extensions, but had no apparent effect on microtubules and other organelles. These effects were reversible. Futhermore, dimethyl sulfoxide, at 0.2-0.4% (concentrations present in 2-4 mug/ml CB medium), had no adverse effect, indicating that the observed abnormalities were direct consequences of CB treatment.

Alcohol dehydrogenase activity in the developing chick embryo.

Before day 9 of incubation, chick embryos contain no measurable alcohol dehydrogenase (ADH) activity. Following day 9 of incubation, chick embryo liver ADH activity increases as a linear function of liver mass. A single dose of ethanol given at the start of incubation is cleared only slowly prior to day 9 of incubation but is completely cleared by day 13. Chick embryo liver ADH has two detectable isozymes throughout development. The percentage contribution of each isozyme to total ADH activity does not change significantly during development. The Km apparent of chick liver ADH is significantly increased shortly after hatching relative to the Km apparent of embryonic ADH. Ethanol exposure during incubation has no effect on the development of ADH activity or isozyme distribution.

Ethanol-induced inhibition of chick brain growth.

Retarded fetal brain growth is associated with a high incidence of mental retardation among the offspring of chronic alcoholic mothers. Research using an embryonic chick model suggests that ethanol exposure suppresses fetal development including suppression of brain growth. Total brain cyclic AMP content and endogenous brain protein kinase specific activity are not altered by ethanol; however, ethanol exposure does significantly stimulate kinase catalytic activity measured in the presence of saturating amounts of exogenous cyclic AMP.

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

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.

The influence of cyclic nucleotides on macromolecular synthesis in cultured neural retina cells.

1. The effect of cyclic nucleotides on aggregates of dispersed embryonic neural retina cells was examined in order to study their influence upon macromolecular synthesis, i.e. protein and DNA. 2. Cyclic AMP, dibutyryl cAMP, cyclic GMP and dibutyryl cGMP were used at various concentrations (5 x 10(-4) -5 mM). 3. The incorporation of labeled precursors into DNA and protein were used to monitor the effect of cyclic nucleotides on cultured aggregates. 4. All nucleotides exhibited a stimulatory effect at 5 x 10(-4) and 5 x 10(-3) mM on macromolecular synthesis, with resulting growth and proliferation of chick neural retina cells. 5. High concentrations (5 x 10(-1) and 5 mM) of cyclic nucleotides exhibited an inhibitory effect upon macromolecular synthesis and a marked cytotoxic effect.

Teratologic effects of LSD in explanted early chick embryos.

Chick embryos were explanted at stage; 4-7 and cultured for 20 h with or without LSD. At any stage 10 mug/ml LSD or higher caused abnormalities in axial structures, particularly somites, in over 50% of the embryos. LSD had no apparent effect on morphogenesis of the heart, but significantly lowered the pulse rate. Cellular degeneration occurred in severely affected structures, but LSD at embryotoxic doses caused alterations in neither cell morphology nor mitotic activity. The effects of LSD were not permanent, i.e., the embryos retained the ability to undergo normal morphogenesis when, after 4-5 h of treatment with 10 mug/ml LSD, they were subcultured on plain nutrient medium.

A single dose of ethanol suppresses rat embryo development in vivo.

In humans and animal models, maternal ethanol consumption during pregnancy results in a variety of fetal defects collectively termed the fetal alcohol syndrome (FAS). Limited follow-up studies suggest that FAS children fail to achieve normal physical or mental development despite significant postnatal intervention. Although the complete FAS appears to result only when chronic, excessive alcohol consumption occurs throughout pregnancy, several investigators have suggested that ethanol consumption at intermediate levels may induce components of FAS. The defect most consistently observed in neonates exposed to ethanol is growth retardation. Even those children whose mothers consume limited amounts of ethanol during pregnancy have a significant incidence of fetal growth deficiency. We now report that a single dose of ethanol administered to female Holtzman rats within 8 hr after mating results in a dose-dependent retardation of cell division in the fertilized ova. The growth retardation is sustained up to 42 hr after the dose and the embryos of young mothers are especially sensitive to ethanol. Animals with high blood alcohol levels (greater than 150 mg/100 ml) show a significant increase in abnormal embryo morphology. These data suggest that maternal consumption of a single dose of ethanol near the time sustained up to 42 hr after the dose and the embryos of young mothers are especially sensitive to ethanol. Animals with high blood alcohol levels (greater than 150 mg/100 ml) show a significant increase in abnormal embryo morphology. These data suggest that maternal consumption of a single dose of ethanol near the time of conception retards embryonic growth and may be detrimental to the developing organism. Further, young female rats receiving a high dose of ethanol had significantly lower uterine weights and a lower number of corpora lutea per ovary, suggesting that a single dose of ethanol has a detrimental effect on maternal reproductive ability.

The effects of indomethacin on fibroblast chemotaxis.

1. Boyden chambers were used to investigate the effects of indomethacin on fibroblast chemotaxis to a conditioned medium. 2. It was determined that indomethacin did not inhibit, but enhanced fibroblast chemotaxis at a concentration of 10(-4) (91%)-10(-6) M (79%). 3. No significant difference was found between controls and cells treated with 10(-8)-10(-10) M indomethacin.

The molecular mechanism of fetal alcohol syndrome (FAS). I. Ethanol-induced growth suppression.

Of the birth defects associated with alcohol consumption during pregnancy, in situ growth retardation resulting in neonates that are small for gestational age is the most common observation in both humans and animal models. A variety of alcohol-induced alterations in maternal, placental and/or fetal physiology have been proposed as the basis for this retarded fetal growth. The molecular mechanism(s) of this retardation, however, is obscure; and it remains to be determined whether the growth suppression is the result of the action of ethanol or its metabolites on embryonic, maternal or placental tissue. Using the embryonic chick as a model which circumvents changes in maternal and placental function, we have measured ethanol-induced growth suppression as a function of embryonic age and ethanol dosage. The data suggest that ethanol per se suppresses the rate of cell division in embryonic tissue resulting in fewer cells/embryo for a given time of gestation. The suppression of cell division is proportional to the ethanol dose and appears to be related to ethanol-induced changes in the metabolism of the prostaglandin hormones and resulting changes in the cyclic-AMP levels of the developing embryos.

The role of extracellular material in chick neurulation. I. Effects of concanavalin A.

The distribution of extracellular material (ECM) in developing neuroepithelium of stage 8+ chick embryos was investigated using lanthanum nitrate. The temporal changes in the distribution of ECM was correlated with the ongoing morphogenetic movements. The most striking finding was that as the folds were about to meet, thick dense ECM appeared between leading edges. Concanavalin A inhibited neural tube closure by binding to cell surfaces and disrupting the usual distribution of ECM in developing neuroepithelium.