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Newborn rats were undernourished from the second postnatal day through 20 days of age and weaned to a diet of laboratory chow ad libitum. Optic nerve development was examined by various light and electron microscopic techniques at 14, 21, 35 and 63 days of age. The degree of undernourishment achieved resulted in body growth lag comparable to results obtained in our previous studies. Although cellularity (cells per photomicrograph area) of the oligodendroglia was unaffected, there was an apparent significant relative reduction in the total number of myelinated fibers by 21 days of age, as determined by light microscopic sampling. Ratios of unmyelinated-to-myelinated fibers were thus estimated by electron microscopy, and results indicated an early increase in the ratio (14 days). Either as the result of catch up in a developmental lag, or as a result of possible restorative effects of rehabilitation, these differences were significant by 35 days of age. The relationship between axon circumference and numbers of myelin lamellae was determined by regression analysis, which revealed a significant reduction in numbers of lamellae over axons of all sizes at 14 days. By 21 days, only fibers in the size range of 1-2 micron of circumference showed a difference, and by 35 days there were no significant differences. These results all indicate that there is a significant myelin reduction in optic nerve of the undernourished rat.

Rats were postnatally undernourished during the suckling period (up to 20 days) and the brainstems of the perfused rats were dissected and prepared for electronmicroscopy at 21, 35 and 63 days of age. The effects on myelin were relatively mild and consisted primarily of a slight reduction in the relative numbers of myelinated fibers, most likely caused by a lag in the rate of loss of non-myelinated fibers, and fewer lamellae in myelinated axons of less than 2.5 micron circumference. Organelles were examined in the interfasicular oligodendroglia and in paragigantocellular reticular neurons immediately dorsal to the pyramidal tract. The numbers of mitochondrial particles in neuronal perikarya were significantly increased by postnatal undernourishment, although the numbers of other organelles appeared normal. Increased numbers of mitochondria persisted in nutritionally rehabilitated rats. Mitochondrial particles in oligodendroglia were not altered.

Rats between 5 and 45 days of age were sacrificed and their sciatic nerves dissected. Myelin was prepared from these sciatic nerves by a procedure involving purification on discontinuous sucrose gradients. The proteins of whole sciatic nerves at different ages and the proteins derived from the myelin isolated from these sciatic nerves were examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Over half of the proteins of sciatic nerve myelin migrated in a single band on the gel (P0). There were only minor changes in the protein distribution of sciatic nerve mylein during development. In contrast, the polyacrylamide gel patterns of whole sciatic nerve homogenate changed markedly during development between 5 and 15 days of age. The amount of P0 protein as a proportion of the total sciatic nerve protein increased from 3% at 5 days of age to 13% at 15 days of age after which it remained constant. Several other proteins which were also characteristic of the isolated myelin increased in relative importance during this time period. Parallel experiments dealing with a metabolic parameter of myelinogenesis, incorporation of intraperitoneally injected [35S]sulfate into sulfatide, were conducted. The maximum synthesis of sulfatide occurred between 6 and 16 days of age, coincident with the marked accumulation of myelin proteins in sciatic nerve.

A double isotope methodology was used to assess the effect of methylxanthine administration on membrane protein synthesis in developing rat brain. Rat pups were given either aminophylline, theophylline, or caffeine in a dosage of 40 mg/kg or 80 mg/kg daily from the second postnatal day through 20 days of age. Results show depressed myelin protein synthesis at 21.24 days by theophylline (80 mg/kg) and caffeine (40 and 80 mg/kg). Synthesis was essentially normal at 27-28 days of age, indicating a possible delay in development followed by a 'catch-up' phenomenon.

We used a double isotope procedure and starved and normal littermate rats to compare relative protein synthesis in the cerebellar nuclear, myelin, synaptosomal, mitochondrial, and microsomal subfractions of postnatally starved animals. The remaining brain tissue was dissected into 6 additional regions (cerebral cortex, medulla oblongata, midbrain, hippocampus, striatum, and hypothalamus) and these were frozen for similar subcellular fractionation and analysis at a later date. The microsomal fraction derived from frozen tissues was discarded. The results show that early postnatal starvation specifically depresses myelin synthesis to about the same extent in all major brain regions at 18 and 21 days of age.

Newborn Long-Evans rats were undernourished by maternal deprivation so that by 20 days of age their body and brain weights were about 45 and 80%, respectively, of the values obtained for control (well-nourished) values. Proteins from myelin of undernourished and control rats were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. At 15 and 20 days of age the proportion of basic and proteolipid protein was reduced in the starved animals relative to controls, indicative of a delay in maturation. However, by 30 days of age the composition of myelin from starved and control animals appeared similar. At all ages the yield of myelin from brains of starved rats was less than 25% of that obtained from control animals. A series of isotope labeling experiments, using a double label design, was carried out to compare relative rates of incorporation of radioactive amino acids into individual proteins of various brain subcellular fractions. In 20-day-old rats the incorporation of [3H] OR [14C] leucine or glycine into myelin proteins, relative to incorporation into proteins of other subcellular fractions, is preferentially depressed (about 60%) in starved animals. Synthesis of all the myelin proteins was depressed, supporting the hypothesis that the high molecular weight proteins isolated with myelin are true myelin constituents. Similar experiments were conducted using [3H]-and [14C] acetate, choline, or glycerol as precursors of lipids. Incorporation of isotope into lipids of myelin, relative to lipids of other subcellular fractions, was also depressed by about 60% in starved animals. In several experiments we studied synthesis during rehabilitation (ad libitum feeding) following 20 days of postnatal starvation. After 6 days of rehabilitation, incorporation of radioactive precursors into myelin, relative to other subcellular fractions, was still depressed. This result was true for both proteins and lipids, and was interpreted as evicence against the initiation of a process leading to a net recovery of myelin (i.e., an irreversible deficit of myelin synthesis is induced by this regime of nutritional deprivation).

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.

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.

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.

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.

Rats undernourished through 17 days of age to produce mild weight lag (less than 20%) had either the same amount, or slightly more myelin than well fed controls. Schedules of undernourishment producing approximately 30, 40 and 50% body weight lags produced corresponding rank-ordered myelin deficits of 25, 55 and 60%, respectively. Brain growth was relatively spared in all cases, never exceeding a deficit of 10%. Absolute myelin deficits did not recover following nutritional rehabilitation, although myelin continued to increase in both normal and all test populations.