Whole eyes reconstituted from embryonic half anlagen: alterations in donor-derived territories in Xenopus pigment chimerae

Grafts from pigmented donor embryonic eye rudiments into albino hosts were used to chart i) fates of local cell groups in three positions in whole eye rudiments, and ii) alterations in graft-derived territories when the posterior half of the rudiment was ablated. Small pigmented patches of graft-derived tissue were conspicuous in albino embryos and tadpoles, enabling us to directly monitor their location and size in the eyes of living animals. The three (right eye) positions marked by pigmented grafts were dorsal (12 o'clock), anterior (3 o'clock), and anteroventral (5 o'clock). Control transplants reared without secondary ablation produced black sector territories in pigment retinal epithelium and iris at corresponding 12 o'clock or 2 o'clock or 4 o'clock positions on the larval eyeball. In the experimental series posterior half-anlagen were ablated. The remaining anterior half-anlagen, each containing a pigmented graft, reconstituted spherical larval eyeballs of reduced size. During healing, donor-derived pigmented sector territories remained coherent, but were altered in position and size compared to controls. Dorsal cells (from 12 o'clock grafts) appeared to move rapidly along the newly formed cut edge into the wound and went on to form expanded black sectors in posterior eye regions. More gradually, sector territories of anterior cells (3 o'clock grafts) and anteroventral cells (5 o'clock grafts) shifted toward dorsal in a counterclockwise direction. Thus all three types of graft derived pigment territories were altered in eye anlage fragments as they healed to form half-size spherical eyes.     

Melanin protects choroidal blood vessels against light toxicity

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.     

Distribution of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in experimental animals studied by postiron emission tomography and whole body autoradiography

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a selective potent neurotoxin which has induced a syndrome similar to parkinsonism both in man and in monkeys. At autopsy degeneration of pigmented nerve cells in the pars compacta of the substantia nigra has been confirmed. The regional distribution of intravenously administered 1-(11C-methyl)-4-phenyl-1,2,3,6-tetrahydropyridine (11C-MPTP) in the brain of Rhesus monkeys was studied by positron emission tomography and the whole body distribution in mice was documented by autoradiography and by impulse counting of selected tissues. A very rapid and high uptake of 11C-MPTP derived radioactivity was seen in areas corresponding to striatum and midbrain, including the substantia nigra area. No elimination from these regions was seen during the study period of 2 h. The uptake was in the order of 7-8 times the homogenous distribution of the radioactivity in the monkey. The uptake was generally high also in other regions of the brain, but there some elimination could be distinguished. Pretreatment of the monkey with spiperone, a selective dopamine receptor antagonist, did not alter uptake nor the kinetics of the 11C-MPTP derived radioactivity. Thus 11C-MPTP does not have a high affinity for postsynaptic dopamine receptors. A remarkably high uptake of 11C-MPTP derived radioactivity was seen in the eye of the monkey. The selective uptake of radioactivity in the eye was also confirmed in pigmented but not in albino mice. The melanin affinity of MPTP may cause high intracellular concentrations of the compound or its metabolites in the melanin containing nerve cells in substantia nigra, which may explain the serious vulnerability of these neurons to MPTP.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.     

Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine

1. The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected intravitreally into the eyes of juvenile (5- to 6-cm) goldfish. 2. Proliferation of rod neuroblasts caused by 6-OHDA (2 micrograms in 2 microliters saline) was detected in retinal wholemounts by immunofluorescence for proliferating cell nuclear antigen (PCNA) 3, 7, 14, 20, or 30 days after injection. 3. The injected dose of 6-OHDA was sufficient to cause permanent loss of dopaminergic interplexiform and serotonergic amacrine cells in the injected eye but not in the contralateral control eye. 4. 6-OHDA increased the density (mm-2) of PCNA-ir cells in the outer nuclear layer (ONL) of the injected eye to 2.65 times the initial density 20-30 days after injection, and it increased the density of PCNA-ir cells in the ONL of the contralateral, untreated eye, equally but after a delay of less than or equal to 7 days with respect to the injected eye. 5. 6-OHDA also increased the density of PCNA-ir cells in the inner nuclear layer (INL) to greater than 20 times the initial density 7 days after injection, followed by a rapid decline almost to control levels by 14 days after injection. 6. The sequence of responses to 6-OHDA, with PCNA-ir cells first scattered in the ONL and then clustered in the INL, suggests that neuroblasts from the ONL migrate to the INL to compensate for toxin-induced cell loss. 7. Double staining for 5-bromodeoxyuridine (BrUdR; a thymidine analogue) and PCNA, carried out on 7 days after intravitreal injection with 6-OHDA, showed that 77% of all PCNA-ir cells in the outer nuclear layer had been in S phase during the previous 24 hr. 8. Immunoreactivity for PCNA was found to be a valid marker for rod neuroblasts which have entered S phase within 1-2 days before sampling and was shown to be especially convenient for investigating the distribution of proliferating cells in whole mounts. 9. In controls injected unilaterally with saline or saline plus 1% dimethyl sulfoxide (DMSO), the differences in densities of PCNA-ir rod precursor nuclei 2-30 days after injection vs. day 0 (uninjected) were statistically insignificant in both injected and uninjected eyes (Negishi et al., 1991). Therefore the local effect of injecting 6-OHDA was due to 6-OHDA itself, not to mechanical damage or nonspecific actions of foreign substances.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice.

The c-locus of the mouse is thought to encode tyrosinase, the key enzyme for melanin synthesis in melanocytes of the skin and the eye. Recently, a mouse cDNA was isolated and shown to confer tyrosine activity on a cell line which expressed no specialized functions for melanin synthesis. To verify that the isolated tyrosinase gene is encoded at the genetically well characterized c-locus, a minigene was assembled from tyrosinase cDNA and tyrosinase genomic DNA and used for generation of transgenic mice. Following microinjection of this construct into fertilized eggs of an albino mouse strain, transgenic mice were obtained which showed pigmentation in skin and eyes. By in situ hybridization, we show expression of the transgene in melanocytes of the hairbulb and in the pigmented cell layers of the eye. We conclude that we have rescued the albino mutation (c/c) by introduction and expression of a functional tyrosinase gene.     

Pharmacokinetic investigation of a 14C-labelled beta 3/alpha tetrapeptide in rats

The solid-phase synthesis and an ADME investigation with albino and pigmented male rats of the doubly 14C-labelled beta/alpha-tetrapeptide derivative Ac-beta3 hTyr-(D)Trp-beta3 hLys-beta3 hThr-lactone (3; Fig. 3) are described. After intravenous (i.v.) and peroral (p.o.) administration of the peptide, its concentration in blood and plasma, its tissue distribution, and the metabolism and the excretion of the peptide were analyzed over a period of up to 7 days post dose. The tetrapeptide in its ring opened form, 5, has a bioavailability of ca. 25%; radioactivity is distributed in the animals in an organ-specific way, and the compound appears to pass the blood-brain barrier to a very small extent, if at all (Tables 1-3 and Figs. 2-6). Excretion (37% renal, 44% fecal, including biliary) of the tetrapeptide 4 days after i.v. administration is almost complete, with only 4.3% remaining in the carcass; 4 days after p.o. administration 97% of the dose has been excreted in the feces. Radiochromatograms taken of plasma (0.5 and 24 h after i.v. dosing) and of urine and feces extracts (0-48 h collected) reveal the presence of lactone 3 and/or the corresponding hydroxy acid 5 with essentially no or very minor other peaks, respectively, representing possible metabolites (Tables 4-6, and Fig. 7 and 8). A comparison with a previous ADME investigation of a beta-nonapeptide show that--except for the lack of metabolism--all aspects of exposure, distribution, and elimination are different (structure-specific properties). The investigated tetrapeptide 3 is a potent and highly specific agonist of the somatostatin receptor hsst4, rendering the results described herein promising for diagnostic and therapeutic applications of beta-peptides.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

Heritable susceptibility to environmentally induced glaucoma in several mutants of Japanese quail

We compared albino (aI), dilute (aID), and wild-type (AI+) quail in their ocular responses to continuous light, the rearing condition that brings on light-induced avian glaucoma (LIAG) in domestic chickens. At age 3 months, all quail kept under 24L/OD showed the retarded corneal growth and corneal flattening characteristic of LIAG. Unlike chickens, quail did not suffer pathological eye enlargement during the early growing period. However, by 6 months of age, 24L/OD albinos showed an almost 20% increase in eye weight compared with 12L/12D albinos. The increase in eye weight for 24L/OD dilutes at 6 months of age was 18%; for 24L/OD wild types, it was 16%. Intraocular pressure, the key criterion for glaucoma, was almost twice as high at 6 months of age in 24L/OD wild types as it was in 12L/12D wild types and showed similar but even greater increases in dilutes and albinos reared under continuous light. Across-genotype comparisons revealed additional effects of the mutant genes themselves: the eyes of albinos were 22.6% larger. The eyes of dilutes showed a similar but smaller response--5% and 6.6%, respectively, and correlated increases in globe dimensional parameters. The flat cornea characteristic of LIAG appeared in all three mutants, but only when environmental light had been kept at 24L/OD. This further separates the LIAG effect from the phenomenon we call albino quail macrophthalmos.     

Development and control of guinea-pig liver estrone sulfate 16 alpha-hydroxylase

Estrone sulfate 16 alpha-hydroxylase activity is undetectable in liver microsomes from fetal guinea-pigs of the English Shorthair variety. Within 2 days of birth, considerable activity is present in both sexes of pigmented and albino animals. In the pigmented group, maximum activity occurs during the second week of life, with the mean values for each of the first 4 weeks, in both sexes, significantly higher than for the corresponding mature (greater than 12-week-old) animals. Immature levels in the albino group are also significantly higher than those of mature albinos. Pigmented females of all ages possess significantly higher activities than do their male counterparts. There are no such sex-related differences in albinos. Pigmented animals of all ages exhibit higher activities than do their albino counterparts. Castration of either sex, pigmented or albino, results in increased enzyme activities as compared with intact or sham-operated controls. Gestation leads to maternal enzyme values which are significantly above those of non-pregnant females, whether pigmented or albino. Beyond the first few days of life, total liver microsomal cytochrome P450 shows no significant change with age, gestation or pigmentation. These data support the conclusion that estrone sulfate 16 alpha-hydroxylase activity in the guinea-pig is markedly diminished, following sexual maturity, by presently unknown factors. This holds for both pigmented and albino animals but the decrease is greater in the latter. This decrease can be reversed by castration in either sex, or by pregnancy and could possibly relate to gonadal-pituitary relationships as demonstrated by others for rat liver hydroxylases.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

Localization of 2-[125I]Iodomelatonin Binding Sites in Mammalian Retina

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40-60% of the binding was specific, as determined with both the agonist 6-chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2-[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2-[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2-[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.     

Effect of oral administration of methadone on hepatic microsomal mixed function oxidase activity in mice

Daily oral administration of methadone (50 mg/kg) brought about a 2-fold increase in the activity of liver N-demethylase in mice within a few days and maintained this high level of activity for the 30-day duration of the experiment. An increase in hepatic microsomal protein and a decrease in pentobarbital sleeping times were noted in the methadone-treated animals. Subcutaneous administration of methadone (20 mg/kg) for 6 days resulted in a much smaller increase in the activity of N-demethylase.     

The metabolism of glucose by the rabbit lens in the presence and absence of oxygen

1. The effect of the absence of oxygen on the metabolism of [U-(14)C]glucose by the rabbit lens has been examined. 2. Protein-free extracts of incubated lens were subjected to electrophoresis followed by chromatography on paper; radioautographs showed 10 compounds substantially labelled. In the absence of oxygen, less radioactivity appeared in glucose, sorbitol, fructose, alanine, glutamic acid, glutathione and nucleotides, and more in lactic acid, alpha-glycerophosphate and glycerol. 3. The radioactivity of lens protein was about two to four times greater after aerobic than after anaerobic incubation. 4. The radioactivity of carbon dioxide was 2- to 4-fold greater after aerobic incubation than after anaerobic incubation. 5. After aerobic incubation of the lens, the radioactivity of all the labelled compounds was higher in the outer part (cortex) than in the inner part (nucleus). 6. Free glycerol has been found to be present in the lens. Its concentration, and that of alpha-glycerophosphate, has been measured in the lens of several species of animal.     

Nuclear transplantation of mouse fetal germ cells into enucleated two-cell embryos

Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F(1) (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.     

Circadian Rhythm of Outside-Nest Activity in Wild (WWCPS), Albino and Pigmented Laboratory Rats

The domestication process of the laboratory rat has been going on for several hundred generations in stable environmental conditions, which may have affected their physiological and behavioural functions, including their circadian system. Rats tested in our ethological experiments were laboratory-bred wild Norway rats (WWCPS), two strains of pigmented laboratory rats (Brown Norway and Long Evans), and two strains of albino rats (Sprague-Dawley and Wistar). Rats were placed in purpose-built enclosures and their cycle of activity (time spent actively outside the nest) has been studied for one week in standard light conditions and for the next one in round-the-clock darkness. The analysis of circadian pattern of outside-nest activity revealed differences between wild, pigmented laboratory, and albino laboratory strains. During daytime, albino rats showed lower activity than pigmented rats, greater decrease in activity when the light was turned on and greater increase in activity when the light was switched off, than pigmented rats. Moreover albino rats presented higher activity during the night than wild rats. The magnitude of the change in activity between daytime and nighttime was also more pronounced in albino rats. Additionaly, they slept outside the nest more often during the night than during the day. These results can be interpreted in accordance with the proposition that intense light is an aversive stimulus for albino rats, due to lack of pigment in their iris and choroid, which reduces their ability to adapt to light. Pigmented laboratory rats were more active during lights on, not only in comparison to the albino, but also to the wild rats. Since the difference seems to be independent of light intensity, it is likely to be a result of the domestication process. Cosinor analysis revealed a high rhythmicity of circadian cycles in all groups.     

The fate of [14C]thalidomide in the pregnant rabbit

1. The fate of [(14)C]thalidomide orally administered to pregnant rabbits at the beginning of the sensitive phase of pregnancy has been studied. 2. After the oral administration of [(14)C]thalidomide on the 192nd hour of pregnancy about 68% of the radioactivity appears in the urine and 22% in the faeces. 3. The urinary (14)C is made up as follows (% of dose): thalidomide (2); alpha-(o-carboxybenzamido)glutarimide (16); 2- and 4-phthalimidoglutaramic acids (11); 2-phthalimidoglutaric acid (0.2); 2- and 4-(o-carboxybenzamido)glutaramic acids and 2-(o-carboxybenzamido)-glutaric acid (29). 4. The plasma (14)C concentration is maximal at 12hr. after dosing and the radioactivity persists for more than 58hr. At 4hr. the main compound in the plasma is thalidomide, but its concentration steadily declines while the concentration of its hydrolysis products increases. 5. At 12, 24 and 58hr. after dosing radioactivity is present in the embryo and the maternal tissues examined. The (14)C concentration in the embryo is at nearly all times higher than that in the plasma, brain, skeletal muscle and fat but lower than that in the liver and kidney. 6. At 4hr. after dosing the mother on the tenth day of pregnancy the specific activities of the embryo and the yolk-sac fluid are similar. 7. Thalidomide is found in the embryo together with seven of its hydrolysis products for more than 24hr. after dosing. The accumulation of radioactivity in the embryo is due to retention of the polar hydrolysis products.     

Experimental data on methicillin tolerance and penetration into the eye media]

Tolerance of methicillin by the eye tissues was studied on its administration subconjunctively, into the front chamber and vitreous body of 20 rabbits. The studies showed that subconjunctival administration of the antibiotic was well tolerated in a dose of 50 mg, and its administration into the front chamber and vitreous body was well tolerated in doses of 1.0-2.5 mg. Penetration of methicillin into the fluids of the front chamber and vitreous body on its instillation into the conjunctive sac in a form of 2.5 per cent solution, its subconjunctival and retrobulbar injection in a dose of 50 mg and intramuscular administration in a dose of 40 mg/kg was studied. Animals with aseptic inflammation of the eyes due to burns of the cornea with 1 N hydrochloric acid were taken into the experiments. The method of agar diffusion with Staph. aureus 209P as the test-microbe was used. The studies showed that the highest methicillin concentrations in the eye media were observed an hour after the antibiotic subconjunctival administration. In the vitreous body they were 16 times lower than those in the front chamber fluid. The retrobulbar injections had no advantages over the subconjunctival administration for the antibiotic maximum concentrations in the vitreous body. The concentration of methicillin in the front chamber fluid on its local administration was many times higher than the minimum inhibitory concentration for staphylococci and may be considered as a therapeutic one.     

Marked accumulation of valproic acid in embryonic neuroepithelium of the mouse during early organogenesis

Valproic acid, an antiepileptic drug, causes neural tube defects in mice and man. 14C-labeled valproic acid (sodium-salt) was administered to pregnant mice on days 8 and 9 of gestation (period of high sensitivity in regard to formation of neural tube defects in this species). Two dose levels of valproic acid (1 and 400 mg/kg) were used; in each case the total radioactivity administered was the same: 400 microCi/kg or 14.7 MBq/kg. Autoradiography combined with computerized densitometry revealed that in low-dose animals most of the radioactivity was confined to maternal liver and kidney, while at high doses more activity was observed in soft tissues and fluids, including amniotic fluid. In the embryo, the neuroepithelium showed the highest concentration, irrespective of dose and survival interval (30 min, 3 h, and 6 h). Upon administration of the high dose, up to five times more radioactivity (approximately 2,000 times more valproic acid) was recovered in embryonic tissues than after the low dose. It is concluded that high doses of VPA saturate the capacities of metabolism, excretion, and protein binding in the maternal organism, resulting in a higher proportion of the dose reaching the embryo, allowing more of the drug to be accumulated by the target organ, the neuroepithelium.     

Affinity of putrescine,spermidine and spermine for pigmented tissues

Determinations of the amounts of putrescine, spermidine and spermine in tissues from mice and cows indicated that the eye melanocytes are very rich in these substances. The concentration of these di-and polyamines was also found to be much higher in pigmented than in albino hair of mice. The melanin polymer has the character of a polyanion — explaining the affinity of these cations for pigmented tissues. Further experiments indicated that these substances to a considerable extent may reach the melanin-containing tissues via the circulation.