Cholesterol turnover in the central nervous system

Immature rat brains were examined for a metabolite of cholesterol which could conceivably form in the central nervous system as a result of cholesterol turnover. A basic assumption was made that an acidic product would be formed, preferentially by oxidative degradation of the cholesterol side chain. Chemical fractionation of brain tissue and thin-layer chromatography of an appropriate acidic fraction indicated the presence of a monohydroxy steroidal acid(s) which remains to be positively identified. Preliminary tracer experiments did not clarify the origin of the unidentified metabolite.     

Effect of extended hypocholesterolemic drug treatment on peripheral and central nervous system sterol content of the rat

Extended treatment of developing or adult rats with a variety of hypocholesterolemic drugs has shown that both the central nervous system (CNS) and peripheral nervous system (PNS) sterol content could be affected in both age groups by this type of treatment. Treatment of developing animals was begun at 5 days of age and continued for 45 days. Adult rats, 300 g at onset, were treated for 35 days. The influence of these drugs on PNS sterol composition has not been previously examined. Diazacholesterol administration caused an accumulation of desmosterol in the CNS and 7-dehydrodesmosterol and desmosterol in the PNS. Zuclomiphene induced a build-up of desmosterol in either tissue. Both of these drugs had a more pronounced effect on developing CNS and PNS than on adult CNS and PNS. Addition of AY-9944 or Triparanol to the zuclomiphene treatment of the developing animals reduced desmosterol accumulation but brought about a build-up of 7-dehydrocholesterol and 7-dehydrodesmosterol.     

Maternal protein deficiency in rat: Effects on central nervous system gangliosides and their catabolizing enzymes in the offspring

Maternal protein deficiency imposed on rats a month prior to conception, and during gestation and lactation, resulted in a significant cell loss in cerebrum, cerebellum, brain stem and spinal cord of pups at weaning. The cerebellum was the most affected central nervous system (CNS) region; it contained only 25% of the normal cell number. Undernourished pups were also found to have a lower concentration of total gangliosides in cerebrum as compared to that of controls. However, the total ganglioside concentration was unaffected in the cerebellum, brain stem and spinal cord by maternal undernutrition. In all regions, undernutrition caused significant changes in the proportions of individual gangliosides; these alterations were region-specific. Sialidase, β-galactosidase, β-glucosidase, and β-hexosaminidase, which are involved in the catabolism of gangliosides, showed higher activities in all the regions of undernourished pups, suggesting that these enzymes may play a role in maintaining the porportions of various ganglioside fractions.     

Effect of prenatal and postnatal exposure to ethanol on rat central nervous system gangliosides and glycosidases

We investigated the effect of maternal alcohol consumption on cell number, gangliosides and ganglioside catabolizing enzymes in the central nervous system (CNS) of the offspring. Virgin female rats of the Charles Foster strain were given 15% (v/v) ethanol in drinking water one month prior to conception and during gestation and lactation. At 21 days postnatal age, the offspring were sacrificed and the brains were separated into cerebrum, cerebellum and brain stem to investigate possible regional variations. Compared to controls, wet weight of cerebrum, cerebellum and brain stem, and of spinal cord was decreased in the pups exposed to alcohol. DNA and protein contents were also found to be lowered in all the CNS regions of the pups exposed to alcohol. Conversely, maternal alcohol consumption was found to increase the concentration and the content of total gangliosideN-acetylneuraminic (NANA) in CNS of the pups. In addition, alcohol treatment was found to induce alterations in the proportions of individual ganglioside fractions. Interestingly, these alterations are somewhat different than those observed in the neonatal brain and spinal cord of the pups subjected to prenatal alcohol exposure. The alterations in the proportions of ganglioside fractions were shown to be region-specific. Maternal alcohol consumption resulted in decreased activities of sialidase, β-galactosidase, β-glucosidase and β-hexosaminidase. The results suggest that the alcohol-associated increases in ganglioside concentration may be at least partly due to the decreased activities of ganglioside catabolizing enzymes.     

The maturation of rat brain myelin

Myelin fractions were prepared from brains of 9- to 90-day-old rats by continuous and discontinuous sucrose density gradient procedures. Total protein and lipid content of myelin showed little variation, but lipid composition changed significantly during maturation. Cholesterol, galactolipids, and ethanolamine glycerophosphatide plasmalogen increased whereas choline glycerophosphatide content decreased with increasing age. The changes in lipid composition were more marked in the myelin prepared by the discontinuous gradient technique. The significance of these lipid changes in relation to their organization in the myelin membrane is discussed. A preliminary report was given at the Federation of Western Societies of Neurological Sciences, March 3–6, 1966, San Francisco, Calif. Career Development Awardee of the National Institute of Mental Health.     

The relationship of biochemical and morphological information in the central nervous system: The problem of sampling

The histology of the central nervous system (CNS) is complex, consisting of membranes derived from a number of cell types as well as specialized membranes associated with structural modifications of the neuron. The cell structure, and thus the membrane content, of the CNS varies with age and from area to area; in no instance is it possible to isolate a single cell type without special microdissection techniques. Neurological disease often involves specific areas of the CNS attacking one or all of the cellular elements in that region. In other instances, damage may be wide-spread, but at the histological level it may be restricted to a single cell type or even to a single membrane. The latter situation is particularly applicable to diseases that attack the myelin sheath. The biochemical investigation of changes in the lipid content of the brain resulting from neurological disease is hampered not only by the morphological complexity of nervous tissue, but by the tendency of destructive processes to be accompanied by proliferation of other cellular elements, both during the acute phase of disease and during the process of repair, which may mask significant abnormalities. Thus, in order to decide which changes in the structural lipids of the diseased brain are meaningful, a knowledge of the histopathology of the disorder is essential. When the morphological changes that accompany a neurological disorder are known, one may choose an appropriate tissue sample for study. Isolation of specific structural or functional subunits of the CNS, dissection of small groups of cells, isolation of single cells and separation of specific membranes are techniques available to sample nervous tissue. These are discussed, and their applicability to the biochemical study of specific neurological diseases is evaluated.     

Molecular species of choline and ethanolamine glycerophospholipids in rat brain myelin during development

The composition of the molecular species of various phospholipid subclasses was examined in myelin isolated from brain of 15-, 21- and 90-day-old rats. The molecular species of diacylglycerophosphocholine (PtdCho), diacylglycerophosphoethanolamine (PtdEtn) and plasmenylethanolamine (PlsEtn) were quantified by high-performance liquid chromatography (HPLC) after phospholipase C treatment and dinitrobenzoyl derivatization. In rat brain myelin, each phospholipid subclass showed a specific pattern of molecular species that changed during development. PtdCho contained large amounts of saturated/monounsaturated and disaturated species and low amounts of saturated/polyunsaturated species. During brain development, the levels of saturated/monounsaturated molecular species increased whereas those of the disaturated and saturated/polyunsaturated species decreased. PtdEtn were characterized by their low levels of disaturated species and a high content of saturated/monounsaturated and saturated/polyunsaturated species, of which those containing fatty acids of the n−3 series decreased, whereas those containing fatty acids of the n−6 series did not change during brain development. The levels of saturated/monounsaturated species increased in PtdEtn. No disaturated molecular species could be detected in PlsEtn. This alkenylacyl subclass contained large amounts of saturated/polyunsaturated, saturated/monounsaturated and dimonounsaturated molecular species. During development, the levels of saturated/polyunsaturated molecular species decreased while those of the two others increased. The data indicated that myelin sheaths undergo phospholipid changes during brain development and maturation.     

Effects of aging on the content,composition and synthesis of sphingomyelin in the central nervous system

Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.5, 21.5 and 26.5-month-old rats. SPH content increased in the cerebral hemispheres, cerebellum and medulla oblongata plus pons as age increased. The highest SPH content was observed in 26.5-month-old rats, with values increasing by 1.74, 2.75 and 0.88-fold, respectively, over 2.5-month-old rats. The SPH fatty acid composition of brains from aged rats was markedly different from that of adult rats. Between 2.5 and 26.5 months of age the monoenoic/saturated fatty acid ratio increased from 0.22, 0.30 and 0.54 to 0.54, 0.68 and 1.03 in cerebral hemispheres, cerebellum and medulla oblongata plus pons, respectively. The percentage and content of fatty acids longer than 22 carbon atoms esterified to SPH increased with age from 18, 26 and 44 to 48, 52 and 62 mole % in cerebral hemispheres, cerebellum and medulla oblongata plus pons in 26.5-month-old rats. In subcortical white matter from aged rats, monoenoic 22–26 carbon atom fatty acids increased more than the saturated ones in 21.5-month-old rats relative to 2.5-month-old rats.In vitro synthesis of SPH from [³H]choline and [³H]palmitic acid in cerebral cortex and cerebellum showed no significant differences between adult rats and those 21.5 months of age. In cerebellum and in cerebral cortex, [¹⁴C] serine incorporation increased in aged rats. The results suggest that aging induces increases in both SPH content and in the monoenoic/saturated fatty acid ratio. These increases are quantitatively different in all brain regions analyzed.     

Alpha tocopherol levels in various regions of the central nervous systems of the rat and guinea pig

The alpha tocopherol contents of various discrete anatomical regions in the central nervous system of adult rats and guinea pigs were assayed using a liquid chromatographic method. All parts of the guinea pig nervous system had lower alpha tocopherol contents per gram wet, dry or lipid weights than the corresponding areas in the rat. In both animals the distribution of alpha tocopherol did not correspond to the distribution pattern of total lipid. There was also a rostral to caudal concentration gradient with respect to alpha tocopherol content; gray matter from cerebral hemisphere has the highest concentration and cervical spinal cord the least. In both animals alpha tocopherol contents per gram dry weight or lipid weight were higher in gray matter areas when compared with white matter areas. The low concentration of alpha tocopherol in spinal cord could make this region more susceptible to damage from deficiency than the rest of the central nervous system.     

Effects of pre- and postweaning undernutrition on polyphosphoinositide pools in rat kidney

The effects of undernutrition on polyphosphoinositide levels in rat kidneys removed and frozen immediately after animal death or 10 min later were determined. Weanling (21-day-old) rats of dams fed a 5 or 22% protein diet and litters fed either normal or protein-deficient diets for an additional six wk were used. Nutritional deprivation lowered phosphatidylinositol-4-phosphate (PtdIns4P) preferentially (35–40%) but but preserved phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P₂) at weaning. This effect was not completely reversed in animals nutritionally rehabilitated after weaning. Postweaning protein deficiency did not reduce the levels of these lipids. Postmortem loss was the same for all five groups, minimal for PtdIns4P and about one-third for PtdIns4,5P₂.     

The effect of conjugated linoleic acid on the antioxidant enzyme defense system in rat hepatocytes

Short-term effects of physiological concentrations of conjugated linoleic acid (CLA) on membrane integrity, metabolic function, cellular lipid composition, lipid peroxidation, and antioxidant enzymes were examined using rat hepatocyte suspension cultures. Incubation with CLA (5–20 ppm) for 3 h decreased the ability of hepatocyte plasma membranes to exclude trypan blue by approximately 25%, and caused leakage of cytosolic lactate dehydrogenase (LDH) into the medium. The significant decrease (P<0.02) in hepatocyte viability as measured by LDH leakage during cell incubation with 10 and 20 ppm CLA was not associated with significant changes in cellular ATP content. Protein synthesis in hepatocytes was elevated (P<0.05) in the presence of 5 and 10 ppm CLA, but at a higher concentration (20 ppm), protein synthesis was similar to that of control cells. Gluconeogenesis was maintained in cells incubated with lower concentrations of CLA (5 and 10 ppm) but was decreased (P<0.02) at the higher concentration. Incubation with 20 ppm CLA for 3 h did not affect the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol synthesis. Both cis-9,trans-11/trans-9,cis-11, and cis-10,trans-12/trans-10,cis-12 isomers of CLA were incorporated to a similar level into hepatocytes. Levels ranged from 3.9 to 4.1%, respectively, of total fatty acids in neutral lipids, and from 0.7 to 0.8%, respectively, of total fatty acids in phospholipids. Cellular lipid peroxidation remained unchanged in the presence of CLA (5–20 ppm), despite significant inhibition (P<0.05) of superoxide dismutase. Catalase activity was maintained near control levels in the presence of 5 and 10 ppm CLA but was significantly decreased in the presence of 20 ppm CLA. Glutathione peroxidase activity was significantly decreased in the presence of 10 ppm CLA. The apparent sensitivity of the antioxidant enzyme defense system of liver cells to CLA, coupled with the lack of effect of CLA on lipid peroxidation in cells, suggests that cytotoxic effects of CLA as described by LDH leakage and decreased gluconeogenesis were not mediated by a prooxidant action in hepatocytes.     

Role of microglia in oxidative toxicity associated with encephalomycarditis virus infection in the central nervous system

The single-stranded RNA encephalomyocarditis virus (EMCV) can replicate in the central nervous system (CNS) and lead to prominent brain lesions in the stratum pyramidale hippocampus and the stratum granulosum cerebelli. Activated microglia cells infected by EMCV produce a massive burst of reactive oxygen species (ROS) via NADPH oxidase 2 (NOX2) activation, leading to neuronal death. Balancing this effect is mechanisms by which ROS are eliminated from the CNS. Cellular prion protein (PrP(C)) plays an important antioxidant role and contributes to cellular defense against EMCV infection. This review introduces recent knowledge on brain injury induced by EMCV infection via ROS generation as well as the involvement of various mediators and regulators in the pathogenesis.     

Closed-loop modeling of central and intrinsic cardiac nervous system circuits underlying cardiovascular control

The baroreflex is a multi-input, multi-output physiological control system that regulates blood pressure by modulating nerve activity between the brainstem and the heart. Existing computational models of the baroreflex do not explicitly incorporate the intrinsic cardiac nervous system (ICN), which mediates central control of heart function. We developed a computational model of closed-loop cardiovascular control by integrating a network representation of the ICN within central control reflex circuits. We examined central and local contributions to the control of heart rate, ventricular functions, and respiratory sinus arrhythmia (RSA). Our simulations match the experimentally observed relationship between RSA and lung tidal volume. Our simulations predicted the relative contributions of the sensory and the motor neuron pathways to the experimentally observed changes in the heart rate. Our closed-loop cardiovascular control model is primed for evaluating bioelectronic interventions to treat heart failure and renormalize cardiovascular physiology.     

Lipid alterations and their reversion in the central nervous system of growing rats deficient in essential fatty acids

Essential fatty acid (EFA) deficiency modifies several biological parameters, i.e., growth, metabolic rate and water balance and induces functional changes in liver, kidney and lung tissues. The brain fatty acid changes reported in the literature are generally smaller than those observed in other tissues. However, EFA deficiency initiated in rats prior to birth and continued for a prolonged period of time results in decreased brain weight, brain lipid and phospholipid content and in considerable changes in polyunsaturated fatty acid distribution, especially in the ethanolamine phosphoglyceride fraction. The unsaturation level of this phospholipid is maintained at a constant level in spite of the fatty acid distribution changes. Brain lipid analyses, carried out at various time intervals in EFA deficient animals, indicate that brain weights are already reduced in 10-day-old deficient rats, that brain phospholipids decrease, especially in the males after three months, and that fatty acid distribution changes begin rather early. The latter consist of a decrease of tetraenes and increase of trienes and of the triene-tetraene ratios as early as at 10 days, a decrease of hexaenes after six months, and after six months, an increase of pentaenes, which are elevated at birth but usually disappear after three months of age. Similar changes are observed in myelin. The induced changes are not completely reversed upon return to normal diet. One of five papers to be published from the Symposium “Lipid Transport,” presented at the AOCS Meeting, New Orleans, April 1970. On leave of absence from the Department of Biochemistry, Medical School of the University of Mississippi, Jackson, Mississippi, Research Career Development Awardee (6-K3-HE-18, 345), National Heart Institute of U.S. Public Health Service.     

The potential role for arachidonic and docosahexaenoic acids in protection against some central nervous system injuries in preterm infants

The risk of central nervous, visual, and auditory damage increases from 2/1000 live births in the normal birthweight to >200/1000 as birthweight falls below 1500 g. Such babies are most likely to be born preterm. Advances in infant care have led to increasing numbers of very-low-birthweight, preterm infants surviving to school age with moderate to severe brain damage. Steroids are one of the current treatments, but they cause significant, long-term problems. The evidence reported here suggests an additional approach to protecting the very preterm infant by supporting neurovascular membrane integrity. The complications of preterm, very-low-birthweight babies include bronchopulmonary dysplasia, retinopathy of prematurity, intraventricular hemorrhage, periventricular leukomalacia, and necrotizing enterocolitis, all of which have a vascular component. Arachidonic acid (AA) and DHA are essential, structural, and functional constituents of cell membranes. They are especially required for the growth and function of the brain and vascular systems, which are the primary biofocus of human fetal growth. Molecular dynamics and experimental evidence suggest that DHA could be the ligand for the retinoid X receptor (RXR) in neural tissue. RXR activation is an obligatory step in signaling to the nucleus and in the regulation of gene expression. Very preterm babies are born with minimal fat stores and suboptimal circulating levels of these nutrients. Postanatally, they lose the biomagnification of the proportions of AA and DHA by the placenta for the fetus. No current nutritional management repairs these deficits. The placental biomagnification profile highlights AA rather than DHA. The resultant fetal FA profile closely resembles that of the vascular endothelium and not the brain. Without this nourishment, cell membrane abnormalities would be predicted. We present a scientific rationale for a common pathogenic process in the complications of prematurity.     

Engineering neural stem cell fates with hydrogel design for central nervous system regeneration

Injuries and disease to the central nervous system (CNS) are accompanied by severe consequences, as the adult CNS has very limited capacity to replace the lost neural cells. Different sources of neural stem cells for CNS tissue regeneration exist, including embryonic stem cells (ESCs), fetal stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs), and so on. However, before stem cell therapy can be a viable option for treatments, many issues still need to be resolved, including low viability, lack of control of stem cell fate, and low cell engraftment after transplantation. Though controlling these parameters is extremely challenging, engineering structures that create permissive niches for the transplanted cells, such as the use of biocompatible hydrogels, is a promising approach. This review will focus on highlighting existing hydrogel systems currently being investigated for CNS tissue regeneration, as well as discuss design criteria for hydrogels and methods for manipulating stem cells within hydrogels systems. Finally, the use of these hydrogel systems as carriers for stem cell transplantation in CNS injury and disease models will be discussed.     

Hydroxy fatty acids from cerebrosides of the central nervous system: GLC determination and mass spectrometric identification

A method has been developed for GLC determination of hydroxy fatty acids (HFA) from cerebrosides of the central nervous system. Identification of HFA present in complex biological mixtures was achieved by the use of the combination gas chromatography— mass spectrometry. GLC separations and mass spectrometric determinations were carried out on the trimethylsilyl derivatives of HFA, which show excellent GLC characteristics and favorable cleavages to determine the position of OH groups on the aliphatic chain. The mass spectra of these derivatives present some unusual rearrangement ions, whose composition has been studied with the aid of deuterium labeled analogs.