Richard C. Wiggins

Brain myelination in the offspring of ethanol-treated rats: in Utero versus lactational exposure by crossfostering offspring of control, pairfed and ethanol treated dams

Pregnant Long-Evans rats were received on day 5 of gestation and divided into 4 treatment groups: (1) 27% calories provided as ethanol in a liquid diet; (2) pairfed i.e., isocaloric liquid diet restricted to match group (1); (3) liquid diet provided ad libitum; and (4) laboratory chow and water provided ad libitum. Litters were culled to 8 pups at birth and crossfostered across dams in all 4 groups to provide offspring falling into 16 different experimental groups, including some exposed to ethanol in utero only and some exposed only during lactation. At birth, blood alcohol levels of dams, culled pups and alcohol levels in the stomach contents of culled pups were measured. All pups were weaned and fed laboratory chow and water ad libitum from 21 days onward. At ages 16, 21, 30 and 52 days, pups were sacrificed, and organ/body weight ratios and brain myelin concentrations were determined. Ethanol treated dams had longer gestational periods. The offspring of ethanol treated dams which were crossfostered to pairfed and well nourished dams during lactation had delayed eye opening, persistent lag in body growth and slightly lower brain myelin concentrations. Offspring of dams which were either pairfed or well nourished during gestation, but crossfostered during lactation to ethanol treated dams, had abnormal organ weights, abnormal brain weights and severely depressed brain myelin concentrations persisting through 52 days of age. Thus, lactational ethanol effects on brain myelin were more severe than gestational effects; body growth was affected more severely by gestational exposure, and gestational effects were generally less severe with adequate nutrition.

Myelin development and nutritional insufficiency

Postnatal undernourishment does not greatly retard the generation of rat brain cells, although there is a slight reduction in total cell numbers and brain size. Possibly the maturation of cells is more severely affected. The ratio of myelinated to non-myelinated fibers is greatly reduced in the corpus callosum and pyramidal tract, and presumably in other areas as well. There is only a slight reduction in the numbers of myelin lamellae for axons of a given size. The recovery of brain myelin and the incorporation of radioactive precursors into purified myelin proteins and lipids are all greatly reduced, leading to a comparatively severe reduction in the brain myelin concentration. The myelin composition is only slightly altered, possibly as a result of delay in its normal chemical maturation. The actual vulnerable period that produces a lasting myelin deficit is the early period that includes oligodendroglia cell proliferation, whereas undernutrition restricted to a later period that includes the actual peak of myelin does not cause a lasting reduction in the brain myelin concentration. The belief that stunting the postnatal proliferation of oligodendroglia largely accounts for the myelin effect has not been substantiated by direct analysis of cell numbers. Consequently, the observed hypomyelination likely results from a failure of oligodendroglia to mature and to initiate myelin formation. The myelin deficit appears largely uniform throughout the brain. Indirect evidence in human studies indicate that comparable effects appear in undernourished infants.

Effect of Reactive Oxygen Species on Myelin Membrane Proteins

Abstract: Fresh myelin, isolated from brainstems of adult rats, was incubated in the presence of Cu2+ and H2O2. Electrophoretic analysis of the reisolated myelin membrane revealed a gradual loss of the protein moiety from the characteristic pattern and an increase in aggregated material appearing at the origin of the gel. The aggregation of proteins was time‐dependent and was concomitant with the accumulation of lipid peroxidation products reactive with thiobarbituric acid. Furthermore, during the course of incubation, there was a gradual decrease in the amount of recovered light myelin and a quantitatively similar increase in heavier myelin subfractions. The aggregation of proteins seems not to be directly related to the buoyant densities of myelin fragments. The peroxidative damage to the myelin proteins may be an important contributor to pathochemistry of myelin sheath, in particular, and in general it implies the susceptibility of the protein moiety of cell membranes to oxygen‐induced deterioration.

The synthesis of myelin and brain subcellular membrane proteins in the offspring of rats fed ethanol during pregnancy

Pregnant Long-Evans rats received either: (1) liquid diet containing 5.15% ethanol; (2) liquid diet pair fed to (1) for total calories; or (3) liquid diet ad libitum. These special diets were administered from the 5th through the 18th days of gestation. Dams received standard laboratory chow and water ad libitum before and after the test interval. Additional dams received standard chow and water throughout the study. Birth weights of offspring in the ethanol group were lower than for offspring of the pair-fed or control groups, and their subsequent growth lagged behind the other groups. Neonate deaths in the ethanol group outnumbered other deaths. Eye opening was delayed, and brain weights appeared low from 16 to 30 days postnatal age, The onset of myelin synthesis was delayed by several days; however, by 30 days of age, the rate of myelin synthesis and net accumulation was comparable to the offspring of pair-fed controls. Thus, the effect of ethanol on brain myelination in the offspring of subject females appears as a delay in myelin initiation and cannot be fully explained by caloric undernourishment. An unexpected observation involved offspring of females fed standard chow throughout the study. The brain myelin concentration in this group was lower than for any of the other groups, which may relate to the higher fat content of the liquids diets and/or the comparatively slow weight gain of pregnant rats on standard chow.

Fetal ethanol exposure: a morphometric analysis of myelination in the optic nerve.

Pregnant Long‐Evans rats were fed a liquid diet containing ethanol during gestation. Controls consisted of both pair‐fed dams and dams fed ad libitum with an equivalent, iso‐caloric diet lacking ethanol. Subsequent effects of ethanol measured in the offspring include a significant lag in the rate at which non‐myelinated axons are lost in association with the initial overproduction of neurons. Additionally, there was a slight lag in the rate of acquisition of myelinated axons; and altogether there was a large increase in the ratio of non‐myelinated to myelinated axons. Frequency spectra of myelinated and non‐myelinated axons by size were normal, and the relationship between axon size and myelin lamellae was also normal. Measured against the dynamic, normal background of rapid cell‐loss and the progressive development of myelin, morphometric demonstration and evaluation of the comparatively small divergences associated with fetal alcohol exposure are difficult; nevertheless, these results are consistent with and help account for the marginal hypomyelination previously observed by quantitative neurochemistry.

Brain maturation following administration of phenobarbital, phenytoin, and sodium valproate to developing rats or to their dams: effects on synthesis of brain myelin and other subcellular membrane proteins.

Abstract: The anticonvulsant drugs phenobarbital, phenytoin, sodium valproate, and phenytoin‐sodium valproate in combination were administered daily to (a) pregnant rats starting on the 5th day after conception, and continued through 17 days postpartum, or (b) to developing rats between 3 and 17 days of age. Each drug was prepared in water and administered at either a therapeutic dose (TD), three times therapeutic dose (3TD), or 9TD. Drug administration had no discernible effect on litter size or sex ratio in the offspring; however, phenobarbital administration to dams caused small but significant reductions in birth weights. Body weights of developing rats treated with anticonvulsant drugs either via dams or directly by intraperitoneal injection lagged behind controls. At 20–24 days of age the brain weights of the offspring of phenobarbital (9TD)‐exposed dams lagged control weights by 5% whereas brain weights in the offspring of the other treated groups were indistinguishable from controls. In contrast, administration of phenobarbital directly to developing rats caused no significant brain weight deficits whereas significant deficits were observed with phenytoin (9TD), sodium valproate (9TD), and phenytoin‐sodium valproate (9TD) in combination. At 20–24 days of age the relative incorporation of radioactive leucine into purified myelin and crude nuclear proteins of drug‐treated rats or the offspring of drug‐treated dams was reduced by 10–20% in all cases. Dose‐related differences were not observed however, and the effects of phenytoin and sodium valproate in combination approximated those of phenytoin administered alone.

The corpus callosum during postnatal undernourishment and recovery: a morphometric analysis of myelin and axon relationships.

This study was designed to compare morphometric relationships between myelin lamellae and axons in undernourished and well nourished developing rats, and in rats nutritionally rehabilitated for two weeks. Although sampling techniques employed in this study were not specifically designed to compare numbers of myelinated fibers in test and control populations, we did observe a trend indicating a reduction in the numbers of myelinated fibers. The mean numbers of myelin lamellae, from an average of all myelinated axons, were not different in control and test population. However, regression analysis of axon sizes by numbers of myelin lamellae revealed significant differences from the normal in 21-day-old undernourished rats. For callosal axons of any size, there were too few myelin lamellae in the undernourished rats. A partial recovery was observed in relatively small fibers by 35 days of age, but no recovery was observed in larger sized fibers. Comparison of the frequency distribution of axon circumferences of myelinated fibers revealed an increase in average axonal caliber. Computation shows that although mean numbers of lamellae were not altered by undernourishment, the axons themselves are increased in size by about 10%. This unexpected result indicates that the relationship normally governing the numbers of myelin lamellae is altered by postnatal nutritional deprivation, and that the relatively larger axon calibers do not produce in the ensheathing oligodendroglia any compensatory increase in the layers of myelin.

Restraint stress during late pregnancy in rats elicits early hypermyelination in the offspring

Female rats were subjected to a regular, daily schedule of 2 hr of restraint stress during the final 6 days of pregnancy. During the first 2 postnatal weeks, adrenal weights were greater than normal in the offspring of the stressed dams. The concentration of brain myelin was higher than control at 14 and 21 days of age but similar to normal by day 40. Early hypermyelination may be partly responsible for early motor development, as previously observed in prenatally stressed rats.

Accumulation and Persistence of Halothane in Adult and Fetal Rat Brain as a Result of Subanesthetic Exposure

Halothane (2-bromo-2-chloro1 , 1 , I-trifluroethane) is a relatively stable and useful anesthetic; however, it is not metabolically inert and consequently there may be undesirable circumstances of exposure. Many investigations have shown various effects of acute exposure to halothane on adult intermediary metabolism and on behavior. Of particular concern are effects of chronic, fetal exposure to trace amounts of halothane, as in the case of unborn offspring of operating room personnel (Van Dyke, 1978; Butcher, 1978). Many of these studies require cautious interpretation as effects are often demonstrated with chronic exposure regimes using doses much higher than actual occupational exposures; however, demonstrated effects include reduced fertility, survival, and growth (Cohen et al., 1971; Corbett, 1972; Bussard et al., 1974; Pope et al., 1978; Wharton et al., 1978), inhibition of DNA synthesis (Grant, 1973; Evenwel et al., 1976; Bandoh and Fujita, 1976; Sturrock and Nunn, 1976), pathological effects (Chang et al., 1974; 1976; Chang and Katz, 1976), decreased brain myelination (Wiggins et al., 1979), and abnormal behavior (Quimby et al., 1974; 1975; Smith et ai., 1978). Because of this current interest in the fetal toxicity of halothane, particularly to the developing nervous system, the present investigation was designed to compare the absorption and retention of halothane in fetal and adult (maternal) brain. We used a subanesthetic concentration (0.5%) that permits routine, accurate detection in tissues. Results show that fetal tissue values are comparable to maternal, and thus neither the interposition of a placenta between the two circulatory systems, nor the absence of detoxification capability in fetal liver has any significant effect on fetal halothane accumulation, or retention.

A comparison of starvation models in studies of brain myelination.

Numerous experimental models have been used to study the relationship between nutritional status and postnatal brain development. Each model has certain advantages and disadvantages in comparison with others. In particular no single model can control for all possible variables of environment and behavior that might conceivably contribute to the experimental result. However, in the present analysis, one can observe that approximately the same effect of postnatal starvation on reducing brain myelination has been observed in three essentially different experimental models.