Kenichirou Inomata

The protective effects of vitamin E on microcephaly in rats X-irradiated in utero: DNA, lipid peroxide and confronting cisternae

Fetuses from rats given either water or 0.03% D,L-alpha-tocopherol acetate (vitamin E) as a drinking fluid and X-irradiated with 100 rad on gestational day 13 were examined on gestational day 21. Mean cerebral weight which was significantly reduced by the X-irradiation was increased by vitamin E supplementation but the level did not reach that in sham-irradiated controls. Administration of vitamin E caused an increase in DNA concentration which was significantly reduced by X-irradiation with water treatment. An increase in the mean level of lipid peroxide formation was observed in the water-treated, X-irradiated group in the sample at zero time but not in the vitamin E-treated, X-irradiated group. In the cytoplasm of fetal cerebral neurons from X-irradiated dams with vitamin E supplementation, confronting cisternae were frequently observed between two nuclear envelopes. Confronting cisternae may be considered as a repair mechanism of vitamin E against X-irradiated neuronal damage in the fetal cerebrum. This study provides evidence of the protection by vitamin E of neuronal development in X-irradiated fetuses, through its antioxidant properties, against attacks by free radicals and/or lipid peroxide.

Fetal alcohol effects: Decreased synaptic formations in the field CA3 of fetal hippocampus

The effects of prenatal ethanol exposure on the synaptic formation in the field CA3 of the hippocampus of fetal rats have been investigated on gestational day 21. Significantly decreased number of synaptic junctions was observed in the fetus showing decreasing cerebral weight either with or without decreasing body weight. The administration of 0.01% zinc with ethanol or 0.02% α‐tocopherol acetate with ethanol during pregnancy resulted in an increased cerebral weight, but did not result in an increased synaptic formation compared to ethanol alone. This result indicates that one of the most vulnerable factors in rat fetus exposed to ethanol in utero is the synaptic formation in the hippocampus. In conclusion, ethanol exposure in utero during a period of brain development roughly equivalent to the first and second human trimesters can produce consistent dysforming effect of synapses and may be associated with the functional impairment of the central nervous system in the fetal alcohol effects.

Decreased density of synaptic formation in the frontal cortex of neonatal rats exposed to ethanol in utero

We examined the developing synaptic junctions in the rat frontal cortex in cases of fetal alcohol syndrome, the objective being to determine the synapse‐mental retardation relationship. On day 21 of gestation, the ultrastructural synaptic junction revealed no obvious differences between the ethanol‐exposed and control rats; however, the number of synapses in ethanol‐exposed rats was one third that of the controls. The possible relationship between synaptic density in the frontal cortex and mental development has to be considered.

A cerium method for the ultracytochemical localization of monoamine oxidase activity

SummaryA cytochemical method based on the complex formation between cerous ions and hydrogen peroxide is described for the ultrastructural localization of monoamine oxidase (MAO). First, the residual MAO activity after fixation was measured by a radiochemical assay technique and was found to be sufficiently retained for cytochemical detection. Although the Tris buffer used in the present method was found to be inhibitory to MAO, considerable activity was still retained after fixation and incubation in Tris.MAO activity, detected as precipitates of cerium perhydroxide, was observed in the mitochondrial outer compartment, mitochondrial cristae and perinuclear space of myocardial cells and endothelial cells of rat heart. MAO activity was also found along the plasma membrane of capillary endothelia. Omission of substrate from the incubation medium or pre-incubation with pargyline, a specific MAO inhibitor, drastically reduced the amount of deposits. The present cerium method seems promising because of its reproducibility and the high electron density of the reaction products.

Fetal Alcohol Syndrome in Rats: Conditions for Improvement of Ethanol Effects on Fetal Cerebral Development with Supplementary Agents

As a possible preventative measure for brain dysfunction in the fetal alcohol syndrome, the effect of zinc or vitamin E supplementation together with ethanol on the fetal cerebrum was investigated in rats. Contrary to our previously published data showing the good effect of 0.01% zinc with 30% ethanol on fetuses, the administration of 0.01% zinc with 20% ethanol, 0.03% vitamin E with 20% ethanol or 0.02% vitamin E with 10% ethanol during pregnancy did not result in a good effect on the body and cerebral weights of fetuses on gestational day 21. The development of dendritic branches on frontal cerebral neurons in fetuses was decreased in the order of the control, zinc with ethanol and ethanol groups. The concentration and content of alpha-tocopherol were increased, but those of zinc were not, in the fetal cerebrum with the maternal administration of vitamin E or zinc together with ethanol, respectively. These results suggest that the improvement of the brain function might depend on the deficient agent induced by maternal ethanol ingestion.

Zinc supplementation in ethanol-treated pregnant rats increases the metabolic activity in the fetal hippocampus

As a possible preventative measure for brain dysfunction in the fetal alcohol syndrome, the effect of zinc supplementation together with ethanol on the fetal cerebrum, especially the hippocampus, was investigated. Female rats of an ethanol group and a control group received 30% ethanol and water, respectively. During pregnancy half of both groups received 0.01% zinc together with ethanol or water. Fetuses on gestational day 21 were examined biochemically and ultrastructurally. Administration of zinc with ethanol resulted in an increased cerebral weight and cerebral RNA content compared to administration of ethanol alone. However, these better results did not reach those of controls without ethanol. In the cytoplasm of hippocampal neurons from dams only given zinc with ethanol confronting cisternae were frequently observed between the rough endoplasmic reticulum and the rough endoplasmic reticulum or the nuclear envelope. Although the functional roles of the confronting cisternae were not definite, it is reasonable to conclude that the confronting cisternae were a repair mechanism of zinc against the inhibitory effect on RNA and protein syntheses of ethanol. This study shows evidence of prevention of the brain dysfunction on ethanol treatment.

High-voltage electron microscopy of E-PTA-stained synaptic junctions in the rat frontal cortex.

The E-PTA-stained synaptic junctions in the adult rat frontal cortex were examined with high-voltage electron microscopy (HVEM). Perforated whole synaptic junctions were clearly shown in the stereo image. The E-PTA staining procedure provides a useful marker for studies of the 3-dimensional structure of synaptic junctions by means of HVEM.

Effects of Low-Power Gallium Aluminium Arsenide Diode Laser Irradiation on the Development of Synapses in the Neonatal Rat Hippocampus

The effect of low-power gallium aluminium arsenide diode laser irradiation on the development of synapses was studied in the radiatum layer and the lacunosum-molecular layer of field CA3 of the neonatal rat hippocampus. Neonatal rats were irradiated with a low-power diode laser (830 nm, 60 mW) at two points located above the hippocampi, for 15 s, respectively, twice per day from birth (day 1) to day 5. The mean body weights of the laser-irradiated animals were found to be lower than those of the control animals, the deficit at day 20 being 22.6%. Moreover, the density of synaptic junctions stained by ethanolic phosphotungstic acid per unit area of the radiatum layer and the lacunosum-molecular layer of the neonatal rat hippocampus was significantly reduced at day 20. It was suggested that the low-power diode laser irradiation affected the development of synapses in the neonatal rat brain.

Abnormal movements in brindled mutant mouse heterozygotes: as related to the development of their offspring--biochemical and morphological studies.

As a possible preventive measure for brain dysfunction in Menkes disease, prenatal treatment by maternal administration of zinc, vitamin E and copper was examined in brindled mutant mice. During pregnancy and lactation, female heterozygous mice received 20 ppm zinc or 0.004% alpha-tocopherol acetate (vitamin E) throughout and 6 ppm copper from gestational day 13 in the drinking fluid, ad libitum. The maternal administration of zinc and vitamin E, as antioxidants, or copper resulted in decreased fetal and neonatal death of offspring, especially those of hemizygous males, as compared with the administration of water only. When offspring did not grow, maternal abnormal movements, which comprised rotatory movements of high speed with tremor and ataxia, were frequently observed. In the heterozygotes with abnormal movements, the level of lipid peroxidation in cerebrum and the concentration of copper in kidney were much higher than those in the heterozygotes with normal movement. Morphologically, in cerebellum of the heterozygotes with abnormal movements, the loss of Purkinje cells, abundance of lipofuscin granules and abnormal mitochondria or degenerative bodies of high electron density were frequently observed, as compared with heterozygotes with normal movement. These findings suggest that the development of hemizygous male mice may be influenced by both copper and oxygen radical metabolism.

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

SummaryThe ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.