CHANGES OCCURRING IN THE CHEMICAL COMPOSITION OF THE CENTRAL NERVOUS SYSTEM DURING FOETAL AND POST-NATAL DEVELOPMENT OF THE SHEEP

(1) The chemical composition of the CNS (separated into cerebrum, cerebellum, brain stem and spinal cord) was determined in sheep during foetal and post-natal development and in adults. (2) The spinal cord differed from the remainder of the CNS in growing more after the period studied (50-day-old foetuses to 5-week-old lambs) than before it. This was largely attributable to lipid accumulation. (3) Chemical growth (accumulation of DNA, protein and lipid) proceeded linearly in spinal cord, logarithmically in cerebrum and cerebellum while in brain stem growth was described by a sigmoid function. (4) Fat-free dry matter, protein, total lipid, cholesterol and phospholipid concentrations increased progressively in all parts of the CNS but DNA concentrations changed little. In the cerebrum alone there was an increase in DNA concentration during maturation suggesting an increased cell population. Cholesterol was present predominantly in the free form but esters were detected in foetal tissues from 70 up to 120 days gestation. (5) Cerebroside, the characteristic lipid of myelin, increased in concentration soon after 85 days of gestation, up to which point very low values were recorded, the rate varying according to the region of the CNS examined. Rates of increase in total regional cerebroside content were used to identify periods of myelination and the results suggest that there are two periods of peak activity, one about 20 days before birth and the other at 10-20 days after birth. (6) The composition of lipids added during the two phases of myelination and during maturation were characteristically different. In the spinal cord, lipid analyses best reflect changes in myelin composition.     

CONGENITAL HYPOMYELINOGENESIS (BORDER DISEASE) OF LAMBS: POSTNATAL NEUROCHEMICAL RECOVERY IN THE CENTRAL NERVOUS SYSTEM

Abstract— In a neurochemical study of experimental Border Disease in lambs it was found that the fresh weights of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) from clinically affected lambs were significantly smaller than those of controls at birth but by 20 weeks of age the cerebrum, cerebellum and brain stem had reached near normal weights. The spinal cord was still considerably smaller, however. Clinical symptoms of the disease (muscular spasms and'hairy'birthcoat) had disappeared during this period, accompanied by a regression in the neurochemical abnormalities seen at birth. Thus the deficiency of myelin lipids was partially made up by the rapid deposition of cerebrosides and by 20 weeks differences in the fatty acid composition of this lipid fraction were no longer apparent. Myelin degeneration as indicated by the presence of elevated levels of esterified cholesterol was apparently absent at 20 weeks of age and this was parallelled by a fall in the level of'anti-myelin'antibodies in the sera of affected lambs. The altered distribution of copper in spinal cord myelin seen at birth had also become nearly normal at the end of the period.     

Effects of Congenital Infection of Sheep with Border Disease Virus on Myelin Proteins

Abstract— Border disease (BD) of sheep is caused by a virus in the genus Pestivirus that results in decreased myelination throughout the CMS when acquired congenitally. Pregnant ewes were inoculated with BD virus at 50 days of gestation, and myelin proteins were quantified in several regions of the CNS during prenatal and postnatal development of infected lambs for comparison with age-matched controls. Newborn field-infected lambs were also examined. Myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were measured by densitometric scanning of western blots. Deficiencies in the myelin proteins were detected as early as 116 days of gestation, and the deficiencies of myelin proteins were most pronounced in the cerebellum at all ages examined. PLP and MBP increased from 10–30% of normal in cerebellar white matter at birth to 40–60% of normal at 6 months, suggesting some catch-up in the amount of compact myelin with development. MAG and CNP were between 70 and 80% of control levels in the cerebellum at birth and at 6 months. Similar results were obtained for the corpus callosum and spinal cord of infected lambs, but the deficiencies of myelin proteins were not as great. A common finding in all regions examined was that MBP and PLP were reduced more than MAG and CNP. This is probably explained by a greater deficit of compact myelin, in which MBP and PLP are localized, than of associated oli-godendroglial membranes, in which MAG and CNP are concentrated. Similar results have been obtained in several dysmyelinating mutants, pointing to common factors in virally and genetically caused hypomyelination. Key Words: Border disease—Myelin—Hypomyelination—Development—Sheeo.     

LIPID DEFICIENCY IN THE CENTRAL NERVOUS SYSTEM OF LANDRACE PIGLETS AFFECTED WITH CONGENITAL TREMOR AIII, A FORM OF CEREBROSPINAL HYPOMYELINOGENESIS

—Congenital tremor, type AIII, is characterized by partial agenesis of the white matter of the CNS affecting mainly the spinal cord. The percentage water content of the fresh cord is consistently higher than normal and other parts of the CNS are sporadically affected. The total lipid content (mg/g fresh tissue) is markedly decreased in the cord but brain stem and cerebellum are less severely deficient; the cerebrum is barely deficient. Total amounts of cholesterol, cerebroside and phospholipid (mmol/part) are significantly reduced predominantly in cerebellum, brain stem and cord. Total DNA and protein contents are decreased to a significant extent only in spinal cord. Broadly similar lipid changes are found in fixed tissues. The data are consistent with sub-normal myelination, associated with a deficiency of oligodendrocytes.     

A regional survey of myelin development: some compositional and metabolic aspects

A survey of differences in composition and metabolism of myelin from five areas of the central nervous system was made in brain and spinal cord slices of the rat from 20 days to 20 months postnatal age. Purified myelin from the forebrain areas showed a composition characteristic of immaturity longer than did myelin from the hindbrain and spinal cord. The trend of chemical maturity is in agreement with the anatomical observations that myelination begins in the hindbrain and proceeds rostrally. Myelin recovery per 100-mg slice increased continually from 20 days to 20 months of age, while the uptake of [1-(14)C]acetate into myelin lipid and of [1-(14)C]leucine into myelin protein decreased precipitously with age. Taking into account the continuous increase in myelin during maturation, a calculation was made of the total amount of incorporation of labeled material into lipids or proteins per 100-mg slice for each region at each age. The metabolic characteristics of myelin from the cerebral cortex (including the corpus callosum), the thalamic area, and the cerebellum were very similar, while myelin from brainstem and spinal cord was metabolically more active, especially at the early ages. Synthesis of lipid in the myelin sheath represents about 50% of the lipid synthesis of the whole brain and about 75% of that of the spinal cord. The proportion of myelin-related protein synthesis is much less, probably less than 10% of the protein synthesis occurring in whole brain and about 15% of that in the spinal cord except at early ages.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

BIPHASIC MYELINATION AND THE FATTY ACID COMPOSITION OF CEREBROSIDES AND CHOLESTEROL ESTERS IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE DOMESTIC PIG

Abstract— The chemical composition of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) was determined in 107 pigs at 11 stages of fetal and postnatal development and also in 6 adults. In cerebrum, cerebellum and brain stem, but not in spinal cord, the rate of increase in weight and the rates of change in lipid content slowed down for a period of about 10 days before and after birth. Cholesterol esters and desmosterol were only found in progressively decreasing amounts during the fetal stages of development and together with DNA these were exceptions to the general increases in the tissue concentrations and total amounts of other components during the period studied.
The onset of myelination, as measured by calculated daily increases in tissue contents of cerebroside took place between 70 and 80 days conceptual age and there were two peaks of activity, the first occurring 2 weeks before and the second 3 weeks after birth. Unlike the rate curve for total spinal cord weight the biphasic accumulation of DNA was not synchronous with myelin lipid accretion and the earlier prenatal DNA peak probably denotes proliferation of oligodendrocytes. The two phases of myelination are discussed in relation to an observed generalized pause in development immediately before and after birth.
Fatty acid analysis of cerebrosides indicated that, in spinal cord, chain elongation and desaturation are associated with myelination and continue with increasing activity until maturity. Consequently there was a progressive decrease in the proportion of saturated fatty acids. The fatty acid components of cholesterol esters in the developing pig were shown to be similar to those found during development in the CNS of other species but different from those found in demyelinating conditions.     

Myelin Lipids in the Developing Cerebrum, Cerebellum, and Brain Stem of Normal and Undernourished Children

Abstract: The developmental lipid profiles in the human cerebrum, cerebellum and brain stem are presented, with special reference to galactolipids as myelin markers to trace myelination in the three main parts of the human CNS. A group of undernourished children were also studied to test the vulnerability of myelinogenesis in the different regions of the human brain. Myelination was well advanced in the brain stem with regard to the other brain regions, a fact reflected in the much higher concentration of myelin lipids in the brain stem of the human foetus of 26 weeks of gestational age. The cerebrum, on the other hand, had the lowest galactolipid concentration during the prenatal period, galactolipid levels in the cerebellum being four times higher. From just before the end of gestation the accretion of galactolipids accelerated enormously in the cerebrum, whereas it slowed down considerably in the cerebellum. Consequently, in relation to prenatal levels galactolipids increased most rapidly in the cerebrum, followed by the cerebellum and finally by the brain stem. These regional differences were in clear contrast to data from the rat, as was the finding that only the cerebrum of undernourished children had a galactolipid concentration significantly decreased with respect to normal values. A relationship between the different myelination patterns in the human and the rat and the distinct vulnerability of myelinogenesis in the two species is suggested.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Blood-brain barrier permeability during dopamine-induced hypertension in fetal sheep.

Dopamine is often used as a pressor agent in sick newborn infants, but an increase in arterial blood pressure could disrupt the blood-brain barrier (BBB), especially in the preterm newborn. Using time-dated pregnant sheep, we tested the hypothesis that dopamine-induced hypertension increases fetal BBB permeability and cerebral water content. Barrier permeability was assessed in nine brain regions, including cerebral cortex, caudate, thalamus, brain stem, cerebellum, and spinal cord, by intravenous injection of the small tracer molecule [(14)C]aminoisobutyric acid at 10 min after the start of dopamine or saline infusion. We studied 23 chronically catheterized fetal sheep at 0.6 (93 days, n = 10) and 0.9 (132 days, n = 13) gestation. Intravenous infusion of dopamine increased mean arterial pressure from 38 +/- 3 to 53 +/- 5 mmHg in 93-day fetuses and from 55 +/- 5 to 77 +/- 8 mmHg in 132-day fetuses without a decrease in arterial O(2) content. These 40% increases in arterial pressure are close to the maximum hypertension reported for physiological stresses at these ages in fetal sheep. No significant increases in the brain transfer coefficient of aminoisobutyric acid were detected in any brain region in dopamine-treated fetuses compared with saline controls at 0.6 or 0.9 gestation. There was also no significant increase in cortical water content with dopamine infusion at either age. We conclude that a 40% increase in mean arterial pressure during dopamine infusion in normoxic fetal sheep does not produce substantial BBB disruption or cerebral edema even as early as 0.6 gestation.     

Developmental patterns of aluminum and five essential mineral elements in the central nervous system of the fetal and infant guinea pig

Al is found in the developing conceptus, but little information is available concerning its tissue distribution and its changes in concentration with age. Because Al has affinity for many of the same biological ligands as the essential mineral cations Ca, Mg, Zn, Fe, and Mn, we hypothesized that Al might show a pattern of developmental concentrations that was similar to one or more of these elements in the brain, a major target of Al toxicity. Concentrations of Al, Ca, Mg, Zn, Fe, and Mn were measured in spinal cord, brainstem, cerebellum, and forebrain of guinea pig fetuses on gestation day (GD) 30 and 45, at birth, and on postnatal day (PND) 3, 6, and 12. Dams were fed commercial guinea pig chow, which contained 47 μg Al/g. Tissue Al and Mn were measured with electrothermal atomic absorption spectrophotometry (ETAAS), and the other elements with inductively coupled axial plasma spectroscopy (ICAP-AES). Al concentrations in the brain regions were highest in spinal cord, brainstem, and cerebellum, and decreased during late gestation and lactation. Al did not show marked increases in regional brain concentrations during the final third of gestation as did Fe, Mg, and Zn. In contrast to Fe and Ca, Al did not accumulate in placenta. Al was the only element to show higher concentrations in spinal cord than in any other tissue at birth. In summary, the tissue distribution of Al did not follow that of essential cations as examined in this study.     

ACTIVITY OF 2'',3''-CYCLIC-NUCLEOTIDE 3''-PHOSPHODIESTERASE IN REGIONS OF RAT BRAIN DURING DEVELOPMENT: QUANTITATIVE RELATIONSHIP TO MYELIN BASIC PROTEIN

2′,3′-Cyclic-nucleotide-3′-phosphodiesterase activity was examined in several regions of rat brain during development, namely optic nerve, olfactory bulb, cerebrum, cerebellum, midbrain, brain stem, and spinal cord. From 4 to 120 days the total activity increased in all regions, although the specific activity approached a constant value in adults. The developmental profile of the enzyme appeared to correlate with the onset of myelination and with the levels of myelin basic protein as well as the appearance of galactocerebroside sulfotransferase. A correlation coefficient of 0.91 was found between total basic protein, expressed as the per cent of the adult (120 day) value, and total enzyme activity over 12–42 days of age (P < 0.001) from six different brain regions as well as for whole brain. By increasing the sensitivity of the assay with the use of [³H-8]adenosine 2′,3′-cyclic monophosphate, we were able to detect activity at birth in both whole brain and spinal cord.     

MYELIN SYNTHESIS IN VITRO: A COMPARATIVE STUDY OF CENTRAL AND PERIPHERAL NERVOUS TISSUE

Abstract— Brain, spinal cord and sciatic nerve from rats at different ages were incubated for 2 h in a medium containing [¹⁴C]acetate and [¹⁴C]leucine as the precursors for synthesis of lipids and proteins. Myelin was purified from the incubated tissues and the specific and total radioactivites of myelin lipids and protein were determined. The uptake of radioactive precursors decreased with increasing age up to 6 months of postnatal age, the decrease following the same pattern for the three types of myelin. After age 6 months the uptake of the protein and lipid precursors reached a plateau that persisted up to 18 months, the oldest postnatal age studied. The amount of myelin isolated and the total myelin lipids extracted from both the central and peripheral nervous systems increased continuously from age 25 days to 18 months after birth. Consequently we suggest that myelination is a process that continues during the whole life of the rat.
The metabolic activity of peripheral nerve myelin was higher than myelin from the CNS at all ages studied. Although myelination in the sciatic nerve begins before that in brain and spinal cord, the three types of myelin apparently reach maturity at the same age. Lecithin exhibited the highest metabolic activity of the individual myelin lipids at all ages in both the central and peripheral nervous system. The metabolic activity of cholesterol in myelin from the 25-day-old rats was similar to that of lecithin but decreased to very low levels in myelin from the 18-month-old rats.     

Neuroprotective effect of mexiletine in the central nervous system of diabetic rats

Both experimental and clinical studies suggests that oxidative stress plays an important role in the pathogenesis of diabetes mellitus type 1 and type 2. Hyperglycaemia leads to free radical generation and causes neural degeneration. In the present study we investigated the possible neuroprotective effect of mexiletine against streptozotocin-induced hyperglycaemia in the rat brain and spinal cord.30 adult male Wistar rats were divided into three groups: control, diabetic, and diabetic-mexiletine treated group. Diabetes mellitus was induced by a single injection of streptozotocin (60 mg/kg body weight). Mexiletine (50 mg/kg) was injected intraperitoneally every day for six weeks. After 6 weeks the brain, brain stem and cervical spinal cord of the rats were removed and the hippocampus, cortex, cerebellum, brain stem and spinal cord were dissected for biochemical analysis (the level of Malondialdehide [MDA], Nitric Oxide [NO], Reduced Glutathione [GSH], and Xanthine Oxidase [XO] activity). MDA, XO and NO levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group increased significantly, when compared with control and mexiletine groups (P < 0.05). GSH levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group decreased significantly when compared with control and mexiletine groups (P < 0.05).This study demonstrates that mexiletine protects the neuronal tissue against the diabetic oxidative damage.     

Vulnerability of myelin development of the chick to the herbicide 2,4-dichlorophenoxyacetic butyl ester

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b. e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.     

Effect of empty uterine space on birth intervals and fetal and placental development in pigs

A substantial loss of embryos occurs between Days 30 and 40 of pregnancy in the pig under crowded intrauterine conditions, but it is not clear whether this loss affects the growth of adjacent conceptuses. Birth intervals are known to increase with decreasing litter size, but the factors responsible are unknown. Two possibilities are that increased birth weight associated with reduced litter size and the empty uterine space and resulting constricted uterine regions that occur in pigs with small litters may impair piglet delivery. To address these, pregnant gilts were laparotomized on Day 35 of pregnancy and one or two fetuses were manually crushed through the uterine wall on the ovarian or cervical end of each uterine horn to create an empty uterine space behind or in front of the litter of piglets, respectively, in relation to the route of delivery from the uterus. A subset of gilts was slaughtered at 105 days of gestation to confirm that the empty uterine spaces were successfully created and to determine their effects on placental and fetal weights of adjacent conceptuses. At slaughter, the lengths of all externally visible empty constricted regions of the uterus were measured. The uterine horns were opened and the lengths of each placenta were measured from the umbilicus toward the ovary and toward the cervix to assess whether placentas developed symmetrically, and then each fetus and placenta was weighed. Fetal crushing successfully created constricted empty uterine regions on the ovarian and cervical ends of the uterine horns. Ovarian-side placental lengths were greater than cervical-side for conceptuses adjacent to fetuses crushed on the ovarian end of the horn. Cervical-side placental lengths were greater than ovarian-side for conceptuses adjacent to fetuses crushed on the cervical end. Both placental and fetal weights were greater (10% and 6%, respectively, P<0.05) for conceptuses adjacent to crushed fetuses compared to nonadjacent conceptuses. Remaining gilts were farrowed to determine the effect of litter size, average birth weights, and treatment on birth intervals of piglets, which were monitored using 24-h video surveillance. The negative association between number of piglets born alive and average birth interval was confirmed and was not explained by litter size-induced reduction in litter average birth weights. Birth intervals and stillbirth rate did not differ between cervically- and ovarian-treated gilts. These results indicate that conceptus loss on Day 35 of gestation can benefit the growth of adjacent placentas and fetuses, but the benefit is small. Increased average birth weight and the presence of empty uterine space that occurs when litter size is reduced does not fully explain the effect of litter size on birth intervals.     

Importance of Dietary Cholesterol for the Maturation of Mouse Brain Myelin

The effect of nil (control), 1% (CH-l) and 5% (CH-5) dietary cholesterol on the myelination of mouse brain, and its deposition in the heart and liver were investigated during infancy. Swiss Webstar female mice were given formulated diets from early gestation, and their pups were weaned on the same diet as that of the individual mothers up to 60 days after birth. The test diets increased the liver weight and cholesterol content compared to the control even in suckling pups (20 days), but did not significantly influence the heart weight until 60 days. The cholesterol content of the heart was not increased by the CH-l diet throughout the feeding period, but it did increase the mole ratio of major myelin lipids and hastened its maturation. Myelin cholesterol was 10% higher in 20-day-old suckling pups in the CH-5 group compared to the control. Data indicate that dietary cholesterol altered the brain myelination rate of weaning mice, and that the mother’s dietary cholesterol influenced myelination of the suckling pups.     

Importance of dietary cholesterol for the maturation of mouse brain myelin.

The effect of nil (control), 1% (CH-1) and 5% (CH-5) dietary cholesterol on the myelination of mouse brain, and its deposition in the heart and liver were investigated during infancy. Swiss Webstar female mice were given formulated diets from early gestation, and their pups were weaned on the same diet as that of the individual mothers up to 60 days after birth. The test diets increased the liver weight and cholesterol content compared to the control even in suckling pups (20 days), but did not significantly influence the heart weight until 60 days. The cholesterol content of the heart was not increased by the CH-1 diet throughout the feeding period, but it did increase the mole ratio of major myelin lipids and hastened its maturation. Myelin cholesterol was 10% higher in 20-day-old suckling pups in the CH-5 group compared to the control. Data indicate that dietary cholesterol altered the brain myelination rate of weaning mice, and that the mother's dietary cholesterol influenced myelination of the suckling pups.