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.     

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.     

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.     

SPINAL CORD LIPIDS AND MYELIN COMPOSITION IN BORDER DISEASE (HYPOMYELINOGENESIS CONGENITA) OF LAMBS

Spinal cords from clinically affected newborn lambs, each with muscular spasms (‘shaking’) and a ‘hairy’ birth coat, were found to be deficient in DNA and to contain less myelin and various lipid components, suggesting retarded CNS development equivalent to about 124 days conceptual age. Cerebrosides were notably deficient in whole cord and isolated myelin and contained more saturated and less unsaturated fatty acids than normal. The rate of cerebroside synthesis assayed in vitro was enhanced and taken with the very low tissue concentrations this indicated faster cerebroside turnover and a less stable myelin in the spinal cords of lambs affected with Border Disease. Marked decreases in plasmalogen concentrations, the redistribution of phospholipid fractions, the presence of about 8 per cent cholesterol in the esterified form and the characteristic fatty acid composition of these esters strongly suggest that degeneration is concomitant with myelin immaturity. Hypocupraemia, low concentrations of copper in the cerebrum and increased concentrations in spinal cord myelin are additional features of the clinical disease. The latter result may be related to myelin immaturity.     

EFFECT OF ZUCLOMIPHENE ON CHOLESTEROL FORMATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE RAT

Abstract— The effect of zuclomiphene, a hypocholesterolemic agent, on developing rat CNS cholesterol biosynthesis was examined. Sterol content and composition was studied in relation to age in four regions of the CNS, cerebrum, brain stem, spinal cord and cerebellum. Sterol content of all four regions was slightly lower in drug-treated animals than in controls. Brain stem and spinal cord were more susceptible to the effects of zuclomiphene than were cerebrum and cerebellum. Drug treatment resulted in the accumulation of desmosterol and zymosterol (5 x -cholesta-8,24-dien-3β-ol) in all CNS regions. After 15 days of drug treatment, desmosterol constituted more than 50% of the total sterol in the four examined regions. Six to 9% of the total sterol was zymosterol.
Examination by electron microscopy indicated only minimal morphological changes. Occasionally, neuronal membranous cytoplasmic inclusion bodies were evident.     

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.     

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 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.     

NEUROCHEMISTRY OF THE SPINAL CORD IN CONGENITAL TREMOR OF PIGLETS (TYPE AII), A SPINAL DYSMYELTMOGENESIS OF INFECTIOUS ORIGIN

Abstract— In piglets affected with congenital tremor type AII the CNS was not morphologically underdeveloped; spinal cord weight, total DNA content and fat-free dry matter differed little from control values. However the total lipid extractable from affected spinal cords was only about 63% of values established for normal newborn piglets. In particular, the cerebroside content (a myelin-specific lipid) was reduced to about 30% of the 'normal' value. This was parallelled by the results of an in vitro assay of cerebroside synthesis from [³H]galactose which indicated a metabolic impairment. The altered fatty acid profile of isolatcd cerebrosides further suggested a derangement of fatty acid synthesis. Unlike the spinal cords of normal newborn piglets, tissues from affected piglets contained significant amounts of cholesterol esters carrying the characteristic fatty acids associated with demyelination. This implied that the reduced quantities of possibly abnormal myelin were unstable. Abnormal swollen oligodendrocytes were commonly present in the spinal cords of affected piglets and this was consistcnt with the observed impairment of myelin formation.     

Cholesterol Esters of the Human Brain During Fetal and Early Postnatal Development: Content and Fatty Acid Composition

Abstract: The content and fatty acid composition of cholesterol esters of the human brain during development from 13 weeks' gestation up to 26 months of age was studied. The three major brain areas, the forebrain, cerebellum, and the brain stem, were studied separately. The concentration of the esters in each brain region was the highest at the earliest fetal age of 13 weeks and fell during growth. However, transient rises in the concentration were observed, at about birth in the forebrain and at 4–5 months after birth in the cerebellum The peak concentration during the transient period (125–150 μg/g fresh tissue of forebrain and 100–125 μg/g of cerebellum) was similar to the concentrations observed in the two parts respectively during early fetal ages. The brain stem also showed similar transient peak at about a few weeks before birth, but only when the esters were expressed as amount per cell. In absolute terms, a clear transient period was evident in the forebrain between birth and 9 months, while in the cerebellum or the brain stem, the total amount of the esters increased up to about 1 year of age and then remained almost unchanged. The major fatty acids of the esters were palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acid. Most of these fatty acids showed certain changes in relative proportions during development. Thus, in the forebrain, palmitic and oleic acid decreased from about 32% and 40% (weight percentages) at 13–15 weeks of gestation to about 20% and 25% respectively at 26 months of age. During this period, linoleic and arachidonic acid increased from about 3% and S% to about 10% and 24%, respectively. Most of these changes occurred after birth. The cerebellum and the brain stem differed only slightly from the forebrain in either the fatty acid composition or the pattern of the developmental changes in the composition.     

TRANS-SULPHURATION IN PRIMATE BRAIN: REGIONAL DISTRIBUTION OF CYSTATHIONINE SYNTHASE, CYSTATHIONINE AND TAURINE IN THE BRAIN OF THE RHESUS MONKEY AT VARIOUS STAGES OF DEVELOPMENT

—The regional distributions of cystathionine synthase, cystathionine and taurine in the brain of the Rhesus monkey were determined at various stages of foetal and postnatal development. Activity of cystathionine synthase was highest in cerebellum, cortical grey areas and globus pallidus, and lowest in subcortical white matter and corpus callosum. There was no marked change in activity in any area during development from the first-trimester foetus to the juvenile animal. In the brain of the juvenile monkey concentrations of cystathionine were highest in subcortical white matter, corpus callosum, and globus pallidus, and lowest in cortical grey matter. There was a sharp increase in concentration between late foetal life and the first 2 weeks of postnatal life and a subsequent more gradual increase during the next 2 years. Concentrations of taurine were highest in lateral cerebellum and neostriatum and lowest in brain stem areas and spinal cord. During the first 6 months of postnatal life, there was a marked decrease in concentration as the brain matured. The regional distribution of cystathionine in brain suggests that this compound may be synthesized in the perikaryon of the nerve cell and transported down axons into white matter. The changes during development suggest the further possibility that cystathionine may have some relationship to myelin and/or myelination.     

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.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

Contribution of acetoacetate to the synthesis of cholesterol and fatty acids in regions of developing rat brain in vivo

³H₂O and [3-¹⁴C]acetoacetate were injected i.p. into developing rats (5–50 days of age). After 2 h the brains were dissected into 6 parts. The incorporation of ³H and ¹⁴C into total fatty acids and into cholesterol in these 6 parts and in the spinal cord was measured. The data were analysed to evaluate the developmental patterns of the synthesis of fatty acids and cholesterol in various parts of the rat CNS and to compare the contribution of acetoacetate to these processes. Our results indicate (1) a large variation between CNS regions in the rates of lipid synthesis as well as in the developmental patterns; highest activities were found in the spinal cord during the third postnatal week, whereas the activities in cortical areas were much lower during all stages of development; (2) a constant ratio between the amounts of label incorporated into lipid fractions from [3-¹⁴C]acetoacetate and from ³H₂O, indicating that acetoacetate contributes to a similar extent to lipid synthesis in all parts of the developing rat CNS; (3) a similar preference in the use of acetoacetate for cholesterogenesis as compared to lipogenesis in all parts of the CNS of suckling rats; (4) a marked increase of this preference after weaning of the pups.     

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.     

THE EFFECT OF GROWTH AND DEVELOPMENT ON THE PHOSPHOLIPIDS OF THE HUMAN BRAIN

Abstract— The phospholipid content and composition of the different regions of the developing human brain were studied. Brains from 25 fetuses and 9 infants, aged 13 weeks gestation to 26 months, were analysed. The concentration of total lipid-P was highest in the brain stem and lowest in the cerebellum at any age. Compared with the forebrain or brain stem, the cerebellum had a sharp phospholipid growth spurt between 3 months before and 6 months after birth. Before birth, the phospholipid pattern was similar in each part of the brain, with choline phosphoglycerides as the major phospholipid. After birth, the pattern in the brain stem changed further and ethanolamine phosphoglycerides became the dominant phospholipid, while in the other two there was little change. In all parts of the brain the proportion of sphingomyelin increased. The relative proportion of serine and inositol phosphoglycerides remained almost constant throughout the whole period of development. The possible significances of the changes in the phospholipids in neurological development are discussed.     

Codistribution of neurite growth inhibitors and oligodendrocytes in rat CNS: appearance follows nerve fiber growth and precedes myelination

Higher vertebrate CNS myelin and oligodendrocytes in vitro contain membrane-bound surface proteins of 35 and 250 kDa with marked inhibitory properties for neurite growth and for fibroblast spreading. The inhibitory activity is neutralized by monoclonal antibody IN-1, which binds to the inhibitory proteins. IN-1 also neutralizes the nonpermissive substrate properties of adult rat optic nerve explants and spinal cord white matter in vitro, thus suggesting a crucial involvement of these inhibitors in the nonpermissive nature of the adult CNS of higher vertebrates. We have determined time of appearance and distribution of the IN-1-sensitive inhibitory activity in the rat. In the optic nerve, inhibitors appear after the period of axonal growth and before myelination. A similar schedule was found for the spinal cord and for the cerebellum. No IN-1-sensitive inhibitory activity was found outside the CNS or in oligodendrocyte-free regions of the CNS. Where investigated, the distribution of inhibitory oligodendrocytes and of IN-1-sensitive inhibitory activity correlated well. Our data suggest that IN-1-sensitive inhibitory activity in vivo might be an oligodendrocyte-specific property.     

Genetic disruption of the growth hormone receptor does not influence motoneuron survival in the developing mouse

In the rodent central nervous system (CNS) during the five days prior to birth, both growth hormone (GH) and its receptor (GHR) undergo transient increases in expression to levels considerably higher than those found postnatally. This increase in expression coincides with the period of neuronal programmed cell death (PCD) in the developing CNS. To evaluate the involvement of growth hormone in the process of PCD, we have quantified the number of motoneurons in the spinal cord and brain stem of wild type and littermate GHR-deficient mice at the beginning and end of the neuronal PCD period. We found no change in motoneuron survival in either the brachial or lumbar lateral motor columns of the spinal cord or in the trochlear, trigeminal, facial or hypoglossal nuclei in the brain stem. We also found no significant differences in spinal cord volume, muscle fiber diameter, or body weight of GHR-deficient fetal mice when compared to their littermate controls. Therefore, despite considerable in vitro evidence for GH action on neurons and glia, genetic disruption of GHR signalling has no effect on prenatal motoneuron number in the mouse, under normal physiological conditions. This may be a result of compensation by the signalling of other neurotrophic cytokines.