Artifactual presence of β-nerve growth factor in adult mouse brain

Adult male mouse brain extracts were determined to contain 0.7 ng β-nerve growth factor (NGF)/mg total protein in a one-site radioimmunoassay (RIA) and 3.6 ng NGF/mg protein in a competition radioreceptor assay using PC 12 cells. When brain extract were immunoprecipitated with antiserum to NGF prior to use in a radioreceptor assay, no reduction in measured NGF content resulted, whereas immunoprecipitation of submaxillary gland extract reduced the NGF level by 83–100%. The immunoassay and radioreceptor assay were then modified by incubating antisera or PC 12 cells first with brain extract, and then, after washing, with ¹²⁵I-labeled authentic mouse NGF. In the modified assays, no NGF was detected in the extracts, indicating that adult mouse brain does not contain NGF itself but does contain an NGF-binding component that causes false positive results in standard one-site RIAs and competition radioreceptor assays.     

Induction of neurofilament triplet proteins in PC12 cells by nerve growth factor

The localization of neurofilament triplet proteins in PC12 cells grown in the absence of (PC12−) or maintained in the presence of (PC12+) nerve growth factor (NGF) was studied using indirect immunofluorescence and monospecific, immunosorbent purified antibodies to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins. The intensity of immunofluorescent staining of the triplet protein was always greater in PC12+ compared with PC12− cells. Neuritic staining was seen in PC12+ cells with all 3 monospecific antibodies to neurofilament proteins. However, the perikaryal distribution of each of the neurofilament proteins differed in both PC12+ and PC12− ells. Monospecific antibodies to P68 protein yielded a ‘ball-like’ cytoplasmic staining pattern whereas monospecific antibodies to P150 protein stained in a stippled pattern. Monospecific antibodies to P200 on the other hand diffusely stained the perikaryal cytoplasm with very faint but detectable foci of f ‘ball-like’ configurations and stippling. Electron microscopic study of PC12+ and PC12− cells revealed intermediate filaments in the cell bodies of both as well as in the processes of the former. ‘Ball-like’ clusters of such filaments were rarely seen. However, these filaments lacked the three-dimensional organization typical of intact neurofilaments.It is concluded that PC12 cells contain dissociated or incompletely assembled immunoreactive neurofilament triplet proteins and that these proteins can be induced by NGF. The PC12 cells are therefore an attractive model system not only for studies of neuronal differentiation but also for studies of neurofilament metabolism and disorders thereof.     

Cyclocytidine-induced release of nerve growth factor from mouse submandibular glands enhances regeneration of sympathetic fibers in adult mice

The injection of a drug endowed with the property of stimulating alpha-adrenergic receptors, cyclocytidine (Cyclo-C), produces drastic depletion of NGF from the granular convoluted tubules (GCT) of the mouse submaxillary salivary gland and a marked NGF level increase in the bloodstream. The NGF discharged from the gland gains access to the blood. Histological studies, immunohistochemistry, in vitro biological assays and radioimmunoassays gave evidence for the growth response elicited by the endogenously released salivary NGF in intact and surgically axotomized sympathetic ganglia. These results suggest that the mouse salivary NGF displays a biological activity on its target sympathetic nerve cells.     

Glial reaction to nerve growth factor in the regenerating optic nerve of the newt (Triturus viridescens)

Nerve growth factor (NGF), administered as a single intraocular injection at the time of nerve transection, elicited cellular hyperplasia and hypertrophy in the astrocyte-like glial cell population of the regenerating newt (Triturus viridescens) optic nerve at 14 days postlesion. More specifically, there was a significant dose-dependent increase in the number of glial cells in response to various NGF concentrations [2 to 2000 biologic units (BU)], which correlated directly with the dose-dependent rise in the neuronal (regenerating axons) area of 14-day regenerating nerve cross sections. We refer to this phenomenon (i.e., cell hyperplasia) as a NGF dose-dependent glial cell response. Quantitation of the astrocyte-like cell perikaryal area and nuclear area, and calculation of cell: nuclear ratios indicated that cell hypertrophy was elicited by the 2000-BU dose of NGF, but not by lower NGF concentrations (i.e., 2 to 200 BU). Corroborative ultrastructural observations were even more revealing. Not only did electron micrographs verify cellular hypertrophy in the astrocyte-like glial cells, but also they revealed hypertrophy of cell processes and a massive increase in the number of microfilaments in response to the 2000-BU NGF treatment. We speculate that these phenomena may represent an NGF dose-independent glial cell response.     

Effects of injecting antibodies to mouse nerve growth factor into the chick embryo

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8- to 15-day-old chick embryo was followed by an enzyme-linked immunoassay. Normal rabbit antibodies (250 μg) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 μg anti-NGF antibodies were injected (40 ng/g). The concentration of anti-NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3–7 were studied in 10-day-old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti-NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti-NGF-treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000–2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti-NGF IgG into the embryo.     

Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons

This study investigated the effects of exogenous nerve growth factor (NGF) on the survival and differentiation in primary culture of sensory neurons isolated from adult (6 months) and aged (2 years) mice. For neurons prepared from adult mice, a concentration effect was evident during a 2 week culture period: Neuronal counts in cultures supplemented with 25 and 50 ng/ml NGF did not differ significantly from those of control cultures without exogenous NGF or those with anti-NGF included in the culture medium, whereas cultures supplemented with either 100 or 200 ng/ml NGF contained higher numbers of neurons throughout the culture period. Cultures prepared from aged mice contained less neurons than those from adult mice, although those supplemented with 100 ng/ml NGF retained higher neuronal numbers than cultures from aged mice which did not receive exogenous NGF. Neuronal diameters were measured to investigate whether specific subpopulations of neurons were more dependent on NGF; the results indicate that neurons of a medium-larger diameter were more prevalent than cells with a smaller diameter following NGF administration. A shape index was calculated for each culture regimen; with longer culture periods a higher proportion of spindle-shaped neurons was observed. © 1993 Wiley-Liss, Inc.     

Role of low-affinity p75 receptor in nerve growth factor-inducible growth arrest of PC12 cells

Mutant PC12 cell clones (PC84 cells) were obtained by transfection with nerve growth factor (NGF) cDNA. These cells secreted active NGF, extended short processes, and proliferated faster than the parental PC12 cells. These features are of great interest because the parental PC12 cells cease proliferation and extend long processes when transfected with NGF cDNA. PC84 cells expressed a high level of acetylcholinesterase activity and neurofilament M, which indicates that PC84 cells were differentiated. The inhibition of TrkA by K252a diminished the short processes of PC84 cells but had no effect on their fast proliferation. The expression level of TrkA in PC84 cells was comparable to that in PC12 cells; whereas that of another NGF receptor, p75, was significantly lower. These data suggest that the decrease of p75 contributed to the continuous growth of PC84 cells, which was confirmed by suppressing p75 activity of PC12 cells with the antisense oligonucleotide of p75 or with anti-p75 neutralizing antibody. The treated cells did not cease proliferation in the presence of NGF and extended short processes. Our results suggest that NGF signaling via TrkA affects the differentiation characteristics of PC12 cells but that an additional signaling via p75 is necessary for the growth arrest of the cells.     

Otoprotective effects of mouse nerve growth factor in DBA/2J mice with early‐onset progressive hearing loss

As it displays progressive hair‐cell loss and degeneration of spiral ganglion neurons (SGNs) characterized by early‐onset progressive hearing loss (ePHL), DBA/2J is an inbred mouse strain widely used in hearing research. Mouse nerve growth factor (mNGF), as a common exogenous nerve growth factor (NGF), has been studied extensively for its ability to promote neuronal survival and growth. To determine whether mNGF can ameliorate progressive hearing loss (PHL) in DBA/2J mice, saline or mNGF was given to DBA/2J mice of either sex by daily intramuscular injection from the 1st to the 9th week after birth. At 5, 7, and 9 weeks of age, in comparison with vehicle groups, mNGF groups experienced decreased auditory‐evoked brainstem response (ABR) thresholds and increased distortion product otoacoustic emission (DPOAE) amplitudes, the prevention of hair cell loss, and the inhibition of apoptosis of SGNs. Downregulation of Bak/Bax and Caspase genes and proteins in cochleae of mice receiving the mNGF treatment was detected by real‐time PCR, Western blot, and immunohistochemistry. This suggests that the Bak‐dependent mitochondrial apoptosis pathway may be involved in the otoprotective mechanism of mNGF in progressive hearing loss of DBA/2J mice. Our results demonstrate that mNGF can act as an otoprotectant in the DBA/2J mice for the early intervention of PHL and, thus, could become of great value in clinical applications. © 2017 Wiley Periodicals, Inc.     

Evaluation of the addition to corticoids of a growth factor (vasopressin) in the palliative therapy of malignant brain tumours

Abstract

Changes in the clinical condition, CT scans and MRIs of patients with recurrent or inoperable astrocytomas and brain metastasis have been observed following treatment with a combinar tion of vasopressin and corticoids. These changes have not been reported with corticoids alone – at least not over the short time reported for this therapy. Ten cases of grade II astrocytomas, seven cases of grade III or IV astrocytomas and six cases of metastasis of bronchial or breast origin were studied. To explain the results, it is proposed that vasopressin delays the ‘escape’ from the effects of corticoids alone and some facts and theories are recalled to help the reader understand the reasoning behind this explanation.     

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.     

In situ hybridization detection of trka mRNA in brain: Distribution,colocalization with p75NGFR and up-regulation by nerve growth factor

In situ hybridization techniques were used to examine the distribution and the nerve growth factor (NGF) regulation of trkA mRNA in the adult rat brain in order to identify neurons in discrete regions of the brain that may be NGF responsive. In agreement with previous studies, trkA mRNA was detected within cells located in the medial septum (MS), diagonal band of Broca (DBB), and caudate. trkA mRNA was also detected in many other regions of the brain, including the nucleus basalis of Meynert, substantia innominata, paraventricular nucleus of the thalamus, interpeduncular nucleus, prepositus hypoglossal nucleus, vestibular nudei raphe obscuris, cochlear nucleus, sensory trigeminal nuclei, and gigantocellular as well as perigigantocellular neurons in the medullary reticular formation. By combining in situ hybridization detection of trkA mRNA with immunocytochemical detection ofp75^NGFR, it was determined that the vast majority (> 90%) of the trkA mRNA-containing cells detected in the MS and DBB also express p75^NGFR. Likewise, the vast majority of p75^NGFR-IR cells detected in the MS and DBB expressed trkA mRNA. Intracerebroventricular infusions of NGF into the third ventricle adjacent to the preoptic area resulted in a 58% increase in relative cellular levels of trkA mRNA in the horizontal limb of the DBB. These data provide evidence that both p75^NGFR and trkA are expressed by NGF-responsive neurons in the MS and DBB. In addition, we note that areas that contained trkA mRNA and that also have been reported to contain p75^NGFR are areas where high-affinity NGF binding sites have been observed autoradio-graphically, whereas areas that contain either trkA or p75^NGFR alone are areas where no high-affinity NGF binding has been reported. Together, these findings suggest that both trkA and p75^NGFR play an important role in the formation of high-affinity NGF receptors in brain and, furthermore, suggest that NGF may have physiological effects within many regions of the brain outside of the basal forebrain.     

Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells.

The uptake of divalent cations and the intracellular concentration of calcium in PC12 cells were studied by flow cytometric analysis using the calcium-sensitive dye, Fluo-3, under a variety of conditions. In particular the actions of nerve growth factor were analyzed. The data show that nerve growth factor stimulates the uptake of divalent cations and increases the intracellular calcium levels of cells attached to collagen-coated plates. The data further indicate that nerve growth factor-dependent increases in the uptake of divalent cations become less pronounced as the intracellular concentration of calcium increases. Intracellular calcium levels increase upon detachment of the cells from the plates and also with increasing cell density. Studies on the uptake of 45calcium confirmed the influence of intracellular calcium levels on nerve growth factor-stimulated calcium uptake. Thus, the effect of nerve growth factor on the uptake of divalent cations is dependent on the calcium levels in the cells, perhaps explaining why previous studies in this field have provided inconsistent results.     

Higher levels of brain derived neurotrophic factor but similar nerve growth factor in human milk in women with preeclampsia

Children born to mothers with preeclampsia have consistently been suggested to be at risk for cognitive and behavioral disorders in later life. Breastfeeding is said to be associated with better neurodevelopment outcomes. Our earlier studies indicated higher levels of docosahexaenoic acid (DHA) in human milk in women with preeclampsia. DHA is known to regulate the expression of neurotrophins and together they play a vital role in neurodevelopment and cognitive performance. The present study examines the levels of maternal plasma and milk neurotrophins [(nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF)] in women with preeclampsia and compares them with normotensive women who served as controls. Singleton pregnant women diagnosed with preeclampsia (n = 72) and controls (n = 102) were recruited for this study from Bharati Hospital, Pune. Plasma and milk samples were analyzed for NGF and BDNF levels using the Emax Immuno Assay System using promega kits. Maternal plasma NGF and BDNF levels were lower (p < 0.01 for both) in women with preeclampsia as compared to the control women. Milk NGF levels were similar while milk BDNF levels were higher (p < 0.05) in the preeclampsia group as compared to controls. Plasma NGF levels were positively correlated with milk NGF levels in the control group. Our results indicate the differential regulation of milk NGF and BDNF levels in women with preeclampsia. The present study suggests a role for both NGF and BDNF in human milk for postnatal brain development. Further studies need to examine the associations of DHA and BDNF in human milk with cognition at later ages.     

No nerve growth factor response to treatment with memantine in adult rats

Summary. Nerve growth factor (NGF) is the most widely examined neurotrophin in the experimental models of Alzheimers disease (AD) and has been shown to prevent the retrograde degeneration of cholinergic neurons. In this study we examined NGF and cholineacetyltransferase (ChAT) changes in several rat brain regions after excitotoxic lesion of the entorhinal cortex with quinolinic acid and tested the effect of memantine on spatial learning in the radial maze after lesion. We observed a significant increase (+26%, p=0.02) of NGF concentrations in the hippocampus of the lesioned rats when compared to sham-lesioned rats. Chronic treatment with memantine showed no significant effect on the NGF increase in the hippocampus (p=0.72). The ChAT activity was significantly increased in the lesioned rats when compared to controls (+16%, p<0.05) and did not depend on treatment with memantine. In spite of this, memantine improved performance of the radial maze. This indicates that memory improving effects of memantine observed in experimental animals and in clinical studies are probably not related to changes in brain NGF content, whereas the observed NGF increase in the denervated hippocampus is probably trauma-related reflecting impaired retrograde transport of hippocampal NGF.Present address: Solvay Pharmaceuticals BV, Weesp, NiederlandePresent address: Institute of Pharmacology PAN, Cracow, Poland     

Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain

The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuron cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals. Glial cells were negative in adults. The distribution of IGF-I in the developing and mature rat brain supports the proposed roles of this peptide as a neuromodulator and neurotrophic factor.     

Effect of nerve growth factor on C-1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice

The in situ C-1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentration in mice sympathectomized with 6-hydroxy-dopamine (6-OHDA) on postnatal days 4-10. Mice were implanted with 1 x 10(6) disaggregated MNB cells 3 days after termination of 6-OHDA administration. NGF (12-15 micrograms/mouse/day) treatment was initiated at the time of MNB cell implantation and continued until sacrifice of the animal. The time interval between tumor cell implantation and detection of palpable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a decrease in myocardial norepinephrine concentration of 88% compared with vehicle-treated animals as well as a significant reduction in total body and organ weight. Average body, brain, heart, and spleen weights were decreased 31%, 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was significantly increased in neonatally sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in sympathectomized mice receiving exogenous NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of nerve growth factor on regeneration of goldfish optic axons

Axonal outgrowth following a crush of the goldfish optic nerve was enhanced if nerve growth factor (NGF) was administered by intraocular injection or by local application to the lesion site. Various forms of NGF (β, 2.5S and 7S) were effective, producing a 20–40% decrease in the time required for recovery of the startle reaction to a bright light. A corresponding increase in axonal outgrowth was revealed by histological examination of the optic nerves. The effect produced by a single intraocular injection given at the time of the lesion was not further increased by subsequent injections. Up to 14 days after the lesion, the size of the retinal ganglion cell bodies and the incidence of nucleoli detectable by light microscopy were not affected by the NGF treatment.