Growth Factor Antiserum Research Articles

Subpopulations of sympathetic neurones differ in their sensitivity to nerve growth factor antiserum.

Sympathetic postganglionic nerve fibres supplying mesenteric arteries and intrinsic ileal neurones differ in their characteristics of regeneration. Since the latter population of neurones occurs predominantly in prevertebral ganglia, which have been reported to be spared to some extent after treatment with antiserum to nerve growth factor (anti-NGF), we have investigated whether the two populations were differentially sensitive to anti-NGF. Newborn rats were treated daily for the first postnatal week with either anti-NGF or 154 mM NaCl solution. At 4 and 8 weeks of age, the presence of a functional sympathetic innervation to the mesenteric arteries and the gut was determined and correlated with the fluorescence histochemical demonstration of noradrenergic fibres. At both ages, stimulation of extrinsic sympathetic fibres caused an inhibition of gut motility, while the mesenteric arteries completely lacked a sympathetic innervation. Retrograde labelling of nerve cell bodies in control and antiserum treated rats confirmed that the sympathetic neurones supplying the ileal neurones were located in the prevertebral, superior mesenteric and coeliac ganglia and in the splanchnic ganglia lying along the greater splanchnic nerves. By interference from retrograde labelling in control animals, sympathetic neurones supplying the mesenteric arteries were present in all these ganglia, as well as in the thoracic and lumbar paravertebral sympathetic chains. The results suggest that two functionally distinct populations of sympathetic neurones, which overlap considerably in their distributions, are differentially sensitive to the immunological postnatal removal of NGF.

Alteration of the intracellular calcium pool of adipose tissue in spontaneously hypertensive rats. No effect of peripheral immunosympathectomy.

The intracellular calcium pool of adipose tissue was estimated by studying the kinetics of 45Ca efflux from the tissue of 9-week old spontaneously hypertensive (SHR) and normotensive Kyoto-Wistar (NKWR) rats subjected to peripheral immunosympathectomy (nerve growth factor antiserum treatment of newborn rats) and in intact (sham-treated) animals of both strains. The development of hypertension in SHR was prevented by immunosympathectomy, however, both groups, SHR-intact and immunosympathectomized rats showed an increase in the intracellular calcium content of adipocytes. These results indicate that the alteration of the membrane control over intracellular calcium distribution in adipose tissue of SHR cannot be attributed to an enhanced activity of the sympathetic nervous system.

Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish ( Carassius auratus): An in vivo and in vitro analysis

In vitro nerve growth factor (NGF) antiserum (anti-NGF) treatment was found to significantly depress retinal ganglion cell neurite outgrowth in goldfish explant culture. Goldfish retinas, conditioned by a 14-day prior optic nerve crush, demonstrated a significant dose response inhibition of neurite outgrowth if incubated with various concentrations of the antiserum (i.e. concentrations from full strength to 1:100) before explanation for tissue culture. NGF added to the incubation medium containing antiserum partially eliminated the inhibition of neurite outgrowth during the first 4 days of explant culture. Antiserum treatment at the higher concentrations (i.e. full strength and 1:1.5 dilution) caused a cessation of nerve growth from explants between culture days 3 and 4. However, controls at this time still exhibited vigorous neurite outgrowth. In vivo treatment with anti-NGF administered intraocularly at 7 days after optic nerve crush (i.e. 7 DPA) was found to significantly reduce the size and complexity of retinal ganglion cell nucleoli when analyzed morphometrically at 14 DPA. No other cell parameters measured (i.e. cell size, nuclear size, cell/nuclear ratios and mitochondrial, Golgi and RER densities) were found to be affected by the single antiserum treatment.

Nerve growth factor and pancreatic APUD cells.

Pancreatic endocrine cells have been considered APUD cells and been thought to be of neural crest origin. Neonatal rats were passively immunized with nerve growth factor antiserum and the development of neural crest derived superior cervical ganglia was markedly inhibited. The pancreatic content of glucagon, insulin, and somatostatin was unaffected, suggesting that pancreatic A,B, and D cells are under different developmental control than are cells of known neural crest origin.

Retinal ganglion cell response to axotomy and nerve growth factor antiserum in the regenerating visual system of the newt ( Notophthalmus viridescens): An ultrastructural morphometric analysis

One 3.0 mg dose of the nerve growth factor antiserum (anti-NGF) injected into the vitreous chamber of the eye at the time of optic nerve transection elicits significant changes in the normal newt ( Notophthalmus viridescens) retinal ganglion cell body response to axotomy at 7 and 14 days postaxotomy (DPA). Light microscopic observations indicate that anti-NGF treatment significantly reduces the per cent of retinal ganglion cells demonstrating nuclear chromatin reactivity (ie., homogeneous to a more heterogeneous state) from33.36 ± 3.02to22.82 ± 2.98%. In addition, the per cent of retinal ganglion cells demonstrating prominent nucleoli is dramatically decreased from32.08 ± 1.64to18.20 ± 1.79% at 7 DPA. It is also important to note that the number of prominent nucleoli in the 7 DPA group is reduced to such an extent by anti-NGF treatment that the value is not significantly different from that of intact controls. Intact controls will routinely exhibit approximately half the number of prominent nucleoli that are normal for the untreated 7 DPA group. A definite dose-response relationship can be shown to exist between the per cent of nuclear reactive ganglion cells demonstrating prominent nucleoli and various anti-NGF concentrations at 14 DPA. There does not appear to be a dose-response relationship between various anti-NGF concentrations and the per cent of retinal ganglion cells demonstrating nuclear reactivity at 14 DPA. However, the degree of nuclear chromatin reactivity appears to be less at the higher anti-NGF concentrations (ie.,≥ 3.0 mg/eye) at 14 DPA. Electron microscopic morphometric analysis reveals that anti-NGF treatment significantly reduces the cell perikaryal area at 7 and 14 DPA while the nuclear area remains unchanged. Therefore, there is a significant decrease in the cytoplasmic/nuclear ratios at both 7 and 14 DPA in response to anti-NGF treatment which appears more pronounced by 14 DPA. Anti-NGF treatment also significantly reduces the mitochondrial and nucleolar densities, as well as the nucleolar areas of cells at 7 and 14 DPA. There are no significant changes in Golgi field densities in response to anti-NGF treatment.

Hypertrophy in the denervated heart: A comparison of central sympatholytic treatment with 6-hydroxydopamine and peripheral sympathectomy with nerve growth factor antiserum

The effects of widespread destruction of central catecholaminergic structures on systemic hemodynamics, ventricular performance, myocardial hypertrophy and the renin-angiotensin system in developing male spontaneously hypertensive rats of the Okamoto strain (SHR) and control normotensive Kyoto Wistar (WKY) rats were examined and contrasted with the effects of peripheral sympathectomy. Both centrally administered 6-hydroxydopamine (6-OHDA) and nerve growth factor antiserum (NGFAS) prevented the development of hypertension in the spontaneously hypertensive rats but did not affect blood pressure in the control rats. Peripheral vascular resistance remained elevated and cardiac and stroke indexes depressed in both 6-OHDA- and NGFAS-treated spontaneously hypertensive rats despite preservation of normal blood pressure. Ventricular performance was depressed in the sham-treated spontaneously hypertensive rats and was not improved by either treatment. Neither 6-OHDA nor NGFAS treatment prevented the development of left ventricular hypertrophy in the spontaneously hypertensive rats despite the decrease in blood pressure. Plasma renin activity was elevated in the 6-OHDA-treated spontaneously hypertensive rats compared with the sham-treated spontaneously hypertensive control rats but was unaffected by treatment with NGFAS. The data indicate that destruction of central and peripheral noradrenergic structures in the immature spontaneously hypertensive rats prevents the development of hypertension by different physiologic mechanisms but does not alter the suppressed ventricular function, the increased peripheral resistance or the development of myocardial hypertrophy. These results support the concept that increased sympathetic activity is necessary for the development of hypertension in spontaneously hypertensive rats but not for the development of myocardial dysfunction or hypertrophy.

Myocardial hypertrophy and ventricular performance in the absence of hypertension in spontaneously hypertensive rats

Ventricular performance and myocardial hypertrophy were studied in maturing spontaneously hypertensive (SH) and normotensive Kyoto-Wistar (WKY) rats from 4 through 24 weeks of age. The development of hypertension in the SH rat was prevented by treatment of the newborn rat with nerve growth factor antiserum (NGFAS). NGFAS treatment had no effect on the blood pressure of WKY rats. The hearts of 1-day- and 2-week-old SH and WKY rats were also examined for evidence of hypertrophy. Cardiac indices of SH rats, regardless of NGFAS treatment, were similar to those of WKY rats until 12 weeks of age at which time they became significantly lower. Stroke indices of both SH groups were lower than those of the WKY groups at all ages studied. Heart rates, however, were significantly higher in the SH rats during the early hypertensive phase but later declined to control levels. NGFAS treatment, despite its effects on hypertension, did not prevent the development of myocardial hypertrophy. Left ventricular hypertrophy was found in SH rats from age 1 day to 24 weeks regardless of NGFAS treatment. In SH rats 1 day to 8 weeks of age right ventricualr hypertrophy was also noted. Thus myocardial hypertrophy is present in SH rats before or very early in the course of the development of hypertension and when hypertension is prevented by peripheral immunosympathectomy. These data support our earlier conclusion that the development of myocardial hypertrophy is independent of elevated blood pressure and suggest that myocardial hypertrophy may be a manifestation of an underlying myocardial abnormality which could account for the hemodynamic alterations found in SH rats.

Effect of peripheral sympathectomy on left ventricular ultrastructure in young spontaneously hypertensive rats

Administration of nerve growth factor antiserum (NGFAS) to spontaneously hypertensive (SH) rats during the first week of life prevents or ameliorates the development of hypertension but does not prevent development of left ventricular hypertrophy or dysfunction. Morphometric analysis of electron micrographs prepared from subepicardial myocardial cells of animals sacrificed at age 82 days shows that NGFAS treatment significantly reduces the fall in cellular concentration of mitochondria characteristic of untreated SH controls. In both groups myofibrillar concentrations were identical and correspond to literature values for normotensive rats. The results, which are consistent with independent control mechanisms for growth of mitochondria and myofibrils, suggest that in this model (a) NGFAS affects mitochondrial concentration either via its effect on arterial blood pressure and/or via some other, unidentified mechanism, and (b) the decreased mitochondrial concentration in myocardial cells of untreated SH rats need not be causally related to the observed cardiac dysfunction.

Regenerative repair in the severed optic nerve of the newt ( Triturus viridescens): Effect of nerve growth factor antiserum

At 14 days after transection those regenerating newt ( Triturus viridescens) optic nerves receiving anti-nerve growth factor treatment were easily distinguished from regenerating controls. Quantitative analysis revealed that antiserum treatment significantly reduced nerve diameter and cross-sectional areas compared to the control groups. Quantitation from electron microscope montages of nerve cross sections revealed similar results. In addition, antiserum treatment significantly reduced the area of regenerating axon fascicles per nerve cross section compared to the control groups. Most significantly, the mean number of regenerating axons per nerve decreased more than 50% in the antiserum-treated group. Regenerating axons appeared normal in all three groups. Axons were filled with clear cytoplasm containing neurofilaments, neurotubules, and an occasional mitochondrion. Axon density was not significantly different among the three groups and axon diameters were similar from 0.1 to 0.8 μm. Distention of glial cell processes surrounding fascicles of axons and increased intra- and extracellular debris may indicate an alteration of glial cell activity in the antiserum group. Many of the 14-day antiserum-treated nerves have the appearance of an untreated transected optic nerve 6 to 10 days after lesion.

Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy.

The effects of peripheral sympathectomy with nerve growth factor antiserum (NGFAS) on blood pressure, systemic hemodynamics, myocardial function, myocardial hypertrophy, and renin were studied in male spontaneously hypertensive (SH) rats of the Okamoto strain and normotensive control Kyoto-Wistar (WKY) rats. NGFAS prevented the developing of hypertension in the SH rats but did not alter blood pressure in the WKY rats. The NGFAS-treated SH rats developed the same hemodynamic abnormalities as the sham-treated rats, including increased peripheral vascular resistance and depressed cardiac output; Indices of left ventricular performance, including peak flow velocity, stroke power, stroke work, dP/dtmax, and flow acceleration (dF/dt), were diminished in the SH rats compared to the WKY rats. NGFAS treatment further depressed ventricular function in the SH rats, but had little effect on the WKY rats; Plasma renin activity in both the SH and WKY rats was unaffected by NGFAS treatment. Although NGFAS treatment effectively prevented the development of hypertension in the SH rats, it did not influence the development of left ventricular hypertrophy as reflected by increases in left ventricular mass, RNA, DNA, and hydroxyproline content. The data suggest that the development of myocardial hypertrophy and myocardial dysfunction in the SH rat is in part independent of hypertension and plasma renin activity.

Effects of nerve growth factor and nerve growth factor antiserum on tyrosine hydroxylase and dihydropterine reductase activities in rat superior cervical ganglia in vivo.

Effects of nerve growth factor and nerve growth factor antiserum on tyrosine hydroxylase and dihydropterine reductase activities in rat superior cervical ganglia in vivo.

Purification of nerve growth factor antibodies by affinity chromatography.

AbstractPure antibodies to nerve growth factor have been isolated from sheep nerve growth factorantiserum by affinity chromatography using 2.5 S nerve growth factor linked to Sepharose 4B by means of cyanogen bromide. The elution of the antibodies was accomplished either at low pH (pH 2) or by high salt concentration (4.5 wMgC12). The purity of the antibodies was established by SDS‐gel electrophoresis. Their immunological activity was tested by imrnunoprecipitation and their biological activity in a tissue culture assay using embryonic chick dorsal root ganglia.

Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells.

Beginning prenatally and during the first week after birth, there is normally a loss of axons in rat cervical sympathetic trunk. To test the hypothesis that this spontaneous axonal loss represents a natural process whereby an excessive number of immature preganglionic axons in the cervical sympathetic trunk adapts to the neuronal population in the superior cervical ganglion, the number of nerve cells in the superior cervical ganglion was reduced in newborn rats by administration of nerve growth factor antiserum, 6-hydroxy-dopamine or postganglionic anxotomy. Quantitative ultrastructural studies of these animals at later stages of development revealed that, with each method, the number of preganglionic axons and Schwann cells was reduced to nearly one-third of normal. These findings indicate that the superior cervical ganglion plays an important role in the development of the cervical sympathetic trunk. Removal of ganglionic cells causes a retrograde loss of preganglionic fibres. This process probably represents an exaggeration of the normal mechanism for elimination of redundant axons. Because the changes in axonal numbers are associated with similar reductions in the number of Schwann cells, it can also be concluded that postnatal Schwann cell proliferation is influenced by axonal populations.

Cardiac hemodynamics and hypertrophy in nerve growth factor antiserum treated spontaneously hypertensive rats

Cardiac hemodynamics and hypertrophy in nerve growth factor antiserum treated spontaneously hypertensive rats

Hypothalamic self-stimulation in rats following immunosympathectomy or central nerve growth factor antiserum injection.

In Experiment 1, immunosympathectomized rats self-stimulated at a much lower rate on high variable ratio schedules of reinforcement than injected controls. No differences were found for responding on continuous reinforcement or low variable ratio reinforcement schedules. In Experiment 2, a similar reduction in varialbe ratio response rate was found for subjects centrally injected with nerve growth factor-antiserum relative to controls. The results suggest a reduction in central catecholamine levels as a result of antiserum treatment.

Location of an isoproterenol-responsive cyclic AMP pool in adrenergic nerve cell bodies and its relationship to tyrosine 3-monooxygenase induction.

To decide whether adenosine 3':5'-cyclic monophosphate (cyclic AMP) plays a role as a second messenger in the trans-synaptic induction of tyrosine 3-monooxygenase (EC 1.14.16.2), it is desirable to discriminate between neuronal and extraneuronal changes in cyclic AMP concentration. Treatment of newborn rats with nerve growth factor antiserum or 6-hydroxydopamine, leading to destruction of 61-85% of the adrenergic nerve cell bodies in the superior cervical ganglion, led to a decrease in cyclic AMP of only 16-28%. This observation demonstrates that a relatively small portion of cyclic AMP is localized in the adrenergic neurons. However, administration of isoproterenol produced an increase (12-fold) in cyclic AMP only in this neuronal pool. Neither single nor repeated injections of isoproterenol led to induction of tyrosine monoxygenase. This, together with previous observations that experimental conditions leading to induction of the enzyme do not produce significant increases of cyclic AMP in the whole ganglion, is taken as an indication that cyclic AMP is not acting as a second messenger in the trans-synaptic induction of tyrosine monooxygenase in the rat superior cervical ganglion. In the rat adrenal medulla, treatment with reserpine led to both a shortlasting (60-90 min) increase in cyclic AMP and a subsequent induction of tyrosine monooxygenase. However, the increase in cyclic AMP was almost completely prevented (40 compared to 320%) by pretreatment of the rats with propranolol while the induction of tyrosine monooxygenase was not diminished. This observation also argues against an exclusive key-function of cyclic AMP in trans-synaptic induction of tyrosine monooxygenase in the adrenal medulla.

Role of sympathetic fibres and of adrenal medulla in the maintenance of cardio-vascular homeostasis in normotensive and hypertensive rats

The Concept of modulation of cardiovascular system by the autonomie nervous system has been accepted for a long time. However, the efferent sympatheticcomponent of the autonomie nervous system is more dynamically related to the peripheral cardiovascular system by a dense plexus of excitatory fibres distributed to the heart and to all vascular beds. Small arterioles which are crucial to the homeostasis of arterial blood pressure were found to be the most densely innervated segments of the vascular tree (N orberg, 1967). In addition to this network of excitatory fibres, the sympathetic nervous system is also responsible for the control of catecholamine secretion by the adrenal medulla which can also influence the heart and blood vessels by acting on specific receptors to catecholamines localized in these structures. Little is known about the relative contribution of each component of the sympathetic system in the maintenance of normal cardiovascular functions. In numerous previous studies, the effects of sympathectomy were studied after surgical denervation, after treatment with nerve growth factor antiserum or after administration of catecholamine depleting drugs such as reserpine. Most of these techniques however were either non specific to the peripheral fibres or, resulted in an incomplete sympathectomy so that the conclusions reached with these procedures may be questionable. The discovery that 6-hydroxydopamine (6-OH-DA) could specifically and selectively destroy adrenergic nerve fibres (T ranzer and Thoenen, 1967) has given a more specific and more useful tool for the study of the role of the sympathetic nervous fibres. In periphery, 6-OH-DA when given in sufficient amount, causes the degeneration of the great majority of sympathetic fibres without destroying the ganglion cell bodies or the adrenal medulla.

Immunosympathectomy: Late effects on the composition of rat cervical sympathetic trunks and influence on axonal regeneration after crush

Uninjured and crushed cervical sympathetic trunks from immunosympathectomized and control rats 8 months old were compared by quantitative electron microscopic techniques. It was demonstrated that: 1. The unmyelinated axonal population, reduced by neonatal administration of nerve growth factor-antiserum, remains stable at approximately one-third of normal. 2. Median diameters of unmyelinated axons were significantly smaller for nerves from nerve grwoth factor-antiserum-treated animals than for control nerves. 3. The number of myelinated axons in cervical sympathetic trunks from nerve growth factor-antiserum-treated animals was reduced to approximately 1/20 of normal. 4. By 8 months of age, the configuration of Schwann cell complexes in nerves from immunosympathectomized animals had changed; the elongated processes and collagen pockets which were present at earlier stages were less prominent. 5. 2 weeks after crush injury, axonal sprouting was similar for immunosympathectomized and control cervical sympathetic trunks.

Comparative studies on the effect of the nerve growth factor on sympathetic ganglia and adrenal medulla in newborn rats

The effect of nerve factor (NGF) and nerve growth factor antiserum (NGF-AS) on sympathetic ganglia and adrenal chromaffin cells of newborn rats was investigated in a comparative study. In confirmation of previous experiments, NGF had a marked hypertrophic and hyperplastic effect in sympathetic ganglia. These morphological changes were accompanied by a selective induction of tyrosine hydroxylase and dopamine β-hydroxylase. The biochemical correlate with the degenerative changes elicited by NGF-AS in sympathetic ganglia was a reduction of all enzymes studied that are located in adrenergic neurones. In the adrenal medulla neither NGF nor NGF-AS produced any detectable morphological changes. However, both procedures resulted in a statistically significant increase in tyrosine hydroxylase and dopamine β-hydroxylase. The paradoxical finding that both NGF and its antiserum produce the same effect on the adrenal chromaffin cells is most plausibly explained by the assumption that both treatments elicit a stress-induced increase in the activity of splanchnic nerves supplying the adrenal medulla, and to a consequent neurally mediated induction of these key enzymes involved in the synsthesis of the adrenergic transmitter, as they do in adult animals under various experimental conditions that lead to an increased activity of the sympathetic nervous system.

Effects of NGF-antiserum in sympathetic neurons during early postnatal development

Mice at birth and on days 4, 7, and 14 were injected with nerve growth factor antiserum (A-NGF) to determine any changes in the nature of lesions during the early postnatal development of the superior cervical sympathetic ganglion. Correlative light and electron microscopic studies revealed both numerical and morphological changes between the first and second weeks of life. The alterations present in the two younger groups resembled those previously described in the newborn. The three zones were seen in many of the nuclei of affected neurons, resulting from nucleolar segregation. Zones A and B contained RNA and probably represent the pars granulosa and pars fibrosa, respectively, of the nucleolus, while zone C contained DNA, initially forming from the nucleolus-associated chromatin. Lesions were fewer in number and less severe in 7- and 14-day-old mice, but still included similar nucleolar alterations. Zone C differed significantly in structure, however, in that large numbers of vacuoles, many of which contained inclusions of varying form and density, were present throughout its substance. Evidence from 3H-uridine experiments, the similarity of nucleolar alterations to those produced by agents known to block DNA-dependent nucleolar RNA synthesis, and the absence of comparable lesions in the adult strongly suggest that A-NGF produces a block in rRNA synthesis in early neuronal development.