Growth Factor Antiserum Research Articles

Progesterone, But Not Estrogen, Stimulates Vessel Maturation in the Mouse Endometrium

The human endometrium undergoes regular periods of growth and regression, including concomitant changes in the vasculature, and is one of the few adult tissues where significant angiogenesis and vascular maturation occurs on a routine, physiological basis. The aim of this study was to investigate the effects of estrogen and progesterone on endometrial vascular maturation in mice. Endometrial tissues were collected from early pregnant mice (d 1-4) and ovariectomized mice given a single 17beta-estradiol (100 ng) injection 24 h before dissection (short-term estrogen regime) or three consecutive daily injections of progesterone (1 mg) with/without estrogen priming (progesterone regime). Experiments were then repeated with the inclusion of mice treated concurrently with progesterone and either RU486 or a vascular endothelial growth factor-A antiserum. Proliferating vascular mural cells (PVMC) were observed on d 3-4 of pregnancy, corresponding with an increase in circulating progesterone. A significant increase in PVMC and alpha-smooth muscle actin (labels mural cells) coverage of vessel profiles were observed in mice treated with progesterone in comparison to controls; no significant change was noted in mice treated with estrogen or with vascular endothelial growth factor antiserum. RU486 treatment did not inhibit the progesterone-induced increases in PVMC and mural cell coverage, although progesterone-induced changes in endothelial and epithelial cell proliferation were inhibited. These results show that progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium. The work illustrates the relevancy of the mouse model for understanding endometrial vascular remodeling during the menstrual cycle and in response to the clinically important progesterone receptor antagonist RU486.

Pre-weaning carvedilol treatment in spontaneously hypertensive rats

Hypertension in spontaneously hypertensive rats (SHR) has been permanently abolished by aggressive treatment regimens targeted against the sympathetic nervous system and adrenal medulla, initiated during the pre-weaning period (guanethidine and nerve growth factor antiserum combined with either adrenal demedullation or prazosin treatment). To investigate the components of the sympatho-adrenal system involved, we treated pre-weaning SHR with the combined α 1- and β-adrenoceptor antagonist carvedilol (60 mg/kg/day s.c.; postnatal days 1–21). Carvedilol treatment significantly blocked adrenoceptors during the treatment period, delayed development (eye opening), reduced growth, and reduced arterial pressure and heart rate. However, there was only modest attenuation of the subsequent development of hypertension at 10 weeks of age (mean arterial pressure 129.5±1.8 versus 136.1±1.6 mm Hg in vehicle-treated littermates; P<0.05). Thus pre-weaning carvedilol treatment slightly but significantly attenuated the development of SHR hypertension at 10 weeks, suggesting that the profound antihypertensive effects of pre-weaning sympatho-adrenal ablation are attributable to factors other than α 1- and β-adrenoceptor-mediated effects of catecholamines during this period.

Anti-interleukin-3 and anti-nerve growth factor increase neonatal mice survival to reovirus type 3 clone 9 per oral challenge

Reovirus type 3 clone 9 (T3C9) induces lethal encephalitis in neonatal, but not adult mice. Whether host factors that promote the development and/or functioning of nervous and gastrointestinal tissues could modulate the pathogenesis of this enteric virus was examined. The results showed that antibody specific for interleukin-3 or nerve growth factor antiserum, but not anti-interleukin-6 or anti-tumor necrosis factor-α/β increased mice survival to T3C9 and decreased viral titers in nervous tissues early after infection. These data suggest that IL-3 and NGF are involved in the pathogenesis of T3C9 infection in neonatal mice.

Neurotrophic factors and pain.

Neurotrophic factors have been shown to play significant roles in the transmission of physiologic and pathologic pain. Nerve growth factor appears to be particularly important. It is crucial for the development of sympathetic and small fiber sensory neurons that serve as nociceptors. It stimulates the expression and release of neuropeptides involved in pain transmission, and interacts with cellular and molecular mediators of inflammation. Blockade with nerve growth factor antiserum demonstrates the critical role of the growth factor in mediating inflammatory hyperalgesia. Administration of nerve growth factor to rodents results in the rapid onset of hyperalgesia. Although the exact mechanism is unknown, several possibilities have been proposed. In clinical trials for the treatment of Alzheimer disease and peripheral neuropathy, induction of pain has been the major adverse event. When administered intracebrebroventricularly, a dull constant back pain resulted. Subcutaneous injection of nerve growth factor induces injection site hyperalgesia, as well as generalized myalgias and arthralgias. Whether the mechanisms underlying these adverse events are identical to those associated with the hyperalgesia in rodents is unknown. In addition to nerve growth factor, other growth factors, such as brain-derived neurotrophic factor and glial cell-derived neurotrophic factor, may be involved in pain pathways. Their precise roles are still being defined, but evidence suggests that they may have particular relevance to neuropathic pain. Understanding the role all these factors play may change the way we approach the treatment of pain in general, and neuropathic pain in particular.

Endogenous nerve growth factor and neurotrophin-3 act simultaneously to ensure the survival of postnatal sympathetic neurons in vivo

For many years nerve growth factor was the only factor known to influence embryonic and postnatal development of sympathetic neurons. Its deprivation by antibody neutralization or gene mutation results in extensive neuron death. Recently it has been shown that these neurons also require neurotrophin-3 for survival in the late developmental period. Using neurotrophin-3 antiserum to neutralize endogenous factor in newborn rats, our laboratory has shown that extensive numbers of neurons are lost from both pre- and paravertebral ganglia, indicating a continuing requirement for neurotrophin-3. In the present study we sought to determine whether neurons could survive in vivo in the presence of excess amounts of either nerve growth factor or neurotrophin-3 alone. Consistent with previous findings, administration of antiserum to nerve growth factor or neurotrophin-3 to newborn rats for eight days, resulted in an extensive loss of sympathetic neurons. Interestingly, administration of neurotrophin-3 together with nerve growth factor antiserum or nerve growth factor with neurotrophin-3 antiserum, reversed this neuronal loss. However the latter combination was less effective than the former. Furthermore, the ability of exogenous nerve growth factor to increase both the number and size of sympathetic neurons was prevented by the simultaneous deprivation of endogenous neurotrophin-3. Unlike nerve growth factor, exogenous neurotrophin-3 failed to rescue the naturally occurring neuronal death in these newborn rats. Further evidence for a physiological role for both nerve growth factor and neurotrophin-3 was found by the detection of both trkA and trkC immunoreactivity in neurons of the superior cervical ganglion.Taken together, these results suggest that sympathetic neurons do not have an absolute requirement for either nerve growth factor or neurotrophin-3 and that the endogenous supply of either factor alone is insufficient to support neuronal survival postnatally. However, while each factor may play similar roles in the regulation of postmitotic neuronal function, some evidence for distinct functions has been identified.

Human Keratinocytes Express the Three Major Splice Forms of Vascular Endothelial Growth Factor

Vascular endothelial growth factor is a powerful mitogen for endothelial cells, recently reported to be produced by keratinocytes. In the present work, we examined human keratinocytes in primary culture for the splice variants of vascular endothelial growth factor. In situ hybridization revealed that 100% of cultured human keratinocytes expressed mRNA for this cytokine, and analysis by reverse transcriptase-polymerase chain reaction indicated that three species of mRNA were produced. Southern hybridization and size calculations of PCR products revealed mRNA species corresponding to 121, 165, and 189 amino-acid forms of this cytokine. Using a rabbit anti-vascular endothelial growth factor antiserum, we radioimmunoprecipitated two molecular weight forms (approximately 45 and 58 kDa, non-reducing conditions) from keratinocyte culture supernatants. Under reducing conditions, three bands of approximately 15, 20, and 24 kDa appeared, corresponding with the predominant forms of vascular endothelial growth factor described. We propose that secretion of vascular endothelial growth factor by human keratinocytes in vivo sustains angiogenesis during physiologic tissue repair and in pathologic states accompanied by neovascularization.

Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells

Normal human (week 17–20) and rat (E16–17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures. Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype. Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.

Medical futility, treatment withdrawal and the persistent vegetative state.

Why do we persist in the relentless pursuit of artificial nourishment and other treatments to maintain a permanently unconscious existence? In facing the future, if not the present world-wide reality of a huge number of persistent vegetative state (PVS) patients, will they be treated because of our ethical commitment to their humanity, or because of an ethical paralysis in the face of biotechnical progress? The PVS patient is cut off from the normal patterns of human connection and communication, with a life unlike other forms of human existence. Why the struggle to justify ending a life which, it is said, has suffered an irreversible loss of the content of consciousness? Elsewhere, the authors have addressed the ethical controversies and confusion engendered by ambiguous terminology, misuse of medical facts and the differing interpretations of what constitutes 'effective' treatment: in particular, the issue of whether in fact artificial nutrition and hydration is a medical treatment, or simply part of the obligatory care owed to all patients, permanently unconscious or not. In this paper, we intend to argue that recent analyses of medical futility, its meaning and ethical implications, despite an absence of public consensus, permit some tentative re-evaluation of our ethical obligations to the PVS patient.

Administration or deprivation of nerve growth factor during development permanently alters neuronal geometry

We investigated whether the administration or deprivation of a neuronal growth factor during development can permanently alter the dendritic architecture of sensitive neurons. Nerve growth factor (NGF) or NGF antiserum treatment in the first 2-3 postnatal weeks markedly affected the survival, size, and dendritic arborization of mouse sympathetic ganglion cells acutely. Six months after the completion of treatment, the number of surviving neurons, cell body size, and higher order dendritic branching had changed considerably from their values at 3 weeks, suggesting that these parameters remain malleable throughout postnatal life. However, the number of primary dendrites, a fundamental determinant of organization within sympathetic ganglia, was permanently altered by the neonatal treatment protocol. The idea emerging from this study is that NGF influences the elaboration of primary dendrites by sympathetic ganglion cells only during a critical developmental period. In maturity, NGF acts as a "maintenance" factor necessary for normal neuronal function and survival, but neurons lose the capacity to respond with wholesale rearrangements of dendritic architecture.

Production of transferable neuronotrophic factor(s) by human midgut carcinoid tumour cells; studies using cultures of rat fetal cholinergic neurons.

A co-culture system was established between human midgut carcinoid tumour cells and rat fetal cholinergic neurons. In monocultures in serum-free media, only tumour cells survived, while neurons deteriorated. In serum-free co-cultures, neurons displayed outgrowth of neuritic processes. Neurons of neuronal serum-free monocultures thrived if supplemented with conditioned media from tumour cell cultures grown serum-free. This indicates that tumour cells produce transferable growth factor(s) with potent neuronotrophic actions. Immunocytochemical studies indicate that this growth factor resembles nerve growth factor immunologically, since tumour cells were strongly immunoreactive after incubation with a rabbit anti-nerve growth factor antiserum, and furthermore expressed immunoreactive nerve growth factor receptors.

Metabolism and Effects of Epidermal Growth Factor and Related Growth Factors in Mammals*

The initial observations of Stanley Cohen in the 1960s established that EGF induced in vivo effects such as precocious eyelid opening and tooth eruption. Subsequently the actions of EGF have been extensively explored in cell culture systems. The receptor for EGF was characterized as a prototype model for other growth factors and the now extensive in vitro data indicate multiple functions for EGF. Moreover, EGF and EGF receptors have been characterized in many tissues, and EGF has been identified in most body fluids of several mammalian species. Interestingly, neither EGF antibody administration to newborn animals nor passive immunization of pregnant rodents against EGF has caused major deleterious effects (except the delay in epidermal maturation events), as might be expected from the in vitro studies. This is in contrast to the effects of nerve growth factor antiserum in developing rodents. Also, to date, no pathological EGF deficiency disorder has been characterized. However, the EGF family of growth factors appears to be important in mammalian development and function, although the precise roles and significance are not yet clear. Members of the family may have a role in embryogenesis and fetal growth since receptors have been identified in fetal tissues. Available evidence suggests that TGF alpha subserves the growth factor family roles in fetal development. In the developing postnatal animal pro-EGF mRNA, immunoreactive EGF, immunoreactive TGF alpha, and EGF receptors are present in many tissues. EGF also is produced and secreted by the maternal mammary gland, and mammary derived EGF appears to be important in gut development in the neonatal rodent. There is now extensive data to indicate important hormone-EGF interactions. In the postnatal period, thyroid and steroid hormones including retinoic acid have been shown to modulate EGF and/or EGF receptors in several tissues. GH increases EGF binding in liver and increases urine EGF concentrations. Moreover, EGF stimulates secretion of several hypothalamic and pituitary hormones, increases placental production of hCG and human chorionic somatomammotropin, increases adrenal cortisol production, and inhibits testicular, ovarian, and thyroid hormone secretions. As summarized in this review EGF has been implicated in a number of developmental events including palate and skin differentiation, growth of hair follicles, eye opening and tooth eruption, lung maturation, gut and liver growth, and differentiation of neurons. These EGF actions probably are mediated via autocrine, paracrine, and endocrine routes. A role for salivary and urine EGF in the maintenance of adult stomal, gut, and urinary epithelial surface integrity seems likely, although not yet proven.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of coculture with Sertoli cells on steroidogenesis in immature rat Leydig cells

The hypothesis has been advanced that Sertoli cells produce one or more follicle-stimulating hormone (FSH)-dependent paracrine factors which stimulate Leydig cell maturation and steroidogenesis. In an attempt to identify these factors we studied the effect of coculture with Sertoli cells on the steroidogenic capacity of immature Leydig cells. It is demonstrated that coculture, during a period of 6 days, markedly increases the capacity of the Leydig cells to secrete C 21-steroids (progesterone, 17α-hydroxyprogesterone, 20α-hydroxypregn-4-en-3-one) and C 19-steroids (testosterone, androst-4-ene-3,17-dione) in response to stimulation with luteinizing hormone (LH). Pretreatment of the cocultures on days 4, 5 and 6 with low concentrations of gonadotropins further enhances the steroidogenic response to LH. This pretreatment results in an overall increase in steroid output. At low concentrations of Sertoli cells and when short incubation times are used, pretreatment with FSH is clearly more effective than pretreatment with LH. Pretreatment with gonadotropins also results in a disproportionate increase in C 19 output caused by increased conversion of C 21 precursors into C 19-steroids. This effect is also observed in Leydig cell monocultures and is mainly due to LH action on Leydig cells. Finally pretreated cocultures display a selective increase in testosterone output. The latter effect is caused by FSH-dependent conversion of androstenedione into testosterone in Sertoli cells. Pretreated cocultures can be maintained for at least 28 days. During this entire period their basal steroid output increases. Using a two-chamber culture system it is demonstrated that direct cell-cell interactions are not required to observe the stimulatory effects of coculture. One or more diffusible factors are involved and continuous contact with these factors is required to maintain the effect. Immunoneutralization experiments using an insulin-like growth factor (IGF-I) antiserum show that IGF-I is an important permissive factor to maintain steroidogenesis in isolated Leydig cells and in cocultures. Under none of the conditions studied, however, does the antiserum neutralize the stimulatory effect of coculture. It is concluded that the stimulatory effects of coculture on the testosterone output of Leydig cells are complex and that important diffusible mediators still remain to be identified.

Acidic Fibroblast Growth Factor Distribution in Normal Human Eye and Possible Implications in Ocular Pathogenesis

Using a rabbit anti-acidic fibroblast growth factor (anti-aFGF) antiserum, we tried to establish a precise mapping of aFGF localization in normal human ocular structures, from samples obtained by autopsies. Cell cultures of retinal pigment epithelium and ciliary pigment epithelium were also established and immunofluorescence studies were performed after 1 month. Corneal and conjunctival epithelia were strongly positive for anti-aFGF antibodies as well as the subcapsular epithelium of the lens. The cortical fibers were weakly reactive and the lens nucleus negative. A strong intracytoplasmic reactivity was observed in the pigmented and nonpigmented epithelial cells of ciliary processes and pars plana, both ex vivo and in vitro. Retina was brightly positive, mostly in the photoreceptor and plexiform layers. The possible involvement of aFGF in normal eye growth and in various ocular diseases was then discussed.

Nerve growth factor can influence growth of cortex cerebri and hippocampus: Evidence from intraocular grafts

The effects of nerve growth factor and antiserum against nerve growth factor on cortical cholinergic projection areas in the central nervous system and cerebellum were evaluated using intraocular grafts of cortex cerebri, hippocampus and cerebellum in rat hosts receiving injections into the anterior chamber of the eye of nerve growth factor (at transplantation, 5 and 10 days after transplantation) or antiserum to nerve growth factor (every 5 days). The controls received cytochrome c or preimmune serum. Growth of grafts was followed by repeated observations directly through the cornea of the host using a stereomicroscope. Nerve growth factor-treated grafts of cortex cerebri and hippocampus grew significantly smaller as compared to the corresponding control grafts. In one experiment, growth of cytochrome c and saline-treated cortex cerebri was compared and no difference in growth was found. Growth of nerve growth factor-treated cerebellar grafts did not differ significantly from growth of cytochrome c-treated grafts. Morphological analysis using Nissl-staining, anitibodies to glial acidic fibrillary protein to evaluate the degree of gliosis and antiserum to neurofilament as a neuronal marker did not reveal any marked differences between nerve growth factor- and cytochrome c-treated grafts. Cortical grafts receiving anti-nerve growth factor antiserum by injection or by immunizing host rats against nerve growth factor showed similar growth to the controls. Similarly, grafts of fetal hippocampus to rats immunized with nerve growth factor were not significantly different from grafts to host rats immunized with cytochrome c. We conclude that exogenous nerve growth factor affects the development of grafted cortex cerebri and hippocampus. The fact that these cortical areas stop growing earlier in the presence of nerve growth factor without the grafts showing evidence of disturbed glial or neuronal populations compared to control grafts indicates that nerve growth factor acts to induce overall/premature differentiation and maturation. The mechanism for this whether or not it is receptor-mediated and which cells are primarily affected by nerve growth factor is not yet known.

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Characterization of a high‐molecular‐weight protein immunoprecipitated by platelet‐derived growth factor antisera

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.

Effect of sialoadenectomy, treatment with epidermal growth factor (EGF) antiserum and replacement of EGF on the epidermis in mice.

Epidermal growth factor (EGF) was present at a mean concentration of 266 +/- 18 (S.E.M.) pmol/mg wet tissue in the submandibular gland of 3-month-old male mice; it was also present in plasma at a concentration of 364 +/- 149 pmol/l. Sialoadenectomy (removal of the submandibular glands) decreased the plasma EGF content to undetectable levels (less than 16.5 pmol/l), lowered the concentration of EGF in the skin from 1.22 +/- 0.11 to 0.47 +/- 0.08 fmol/mg wet tissue and reduced the thickness of the epidermis from 28.9 +/- 2.7 to 11.0 +/- 0.8 micron in 3 weeks (P less than 0.001). Epidermal growth factor antiserum given to sialoadenectomized mice further decreased the thickness of the epidermis to 8.3 +/- 0.6 micron. No appreciable change was observed in the dermis and subcutaneous tissue. In sialoadenectomized mice, replacement of EGF prevented the decrease in thickness of the epidermis in a dose-dependent manner when started immediately after the operation. Treatment with EGF also effectively restored the normal morphology of the epidermis when its thickness had declined to its lowest level. These results suggest that EGF plays a physiological role in the maintenance of the epidermis.

Epidermal growth factor deficiency during pregnancy causes abortion in mice

The concentration of epidermal growth factor in the submandibular gland and plasma of female mice increased substantially during pregnancy. Pregestational sialoadenectomy (removal of the submandibular glands) attenuated the rise in plasma epidermal growth factor during gestation and reduced the number of mice completing a term pregnancy by approximately 50%. Epidermal growth factor replacement therapy given to these mice during pregnancy improved the outcome of complete pregnancy to 86%. The administration of anti-epidermal growth factor antiserum in a daily dose of 50 or 100 μl to sialoadenectomized pregnant mice increased the percentages of abortion to 67 and 100, respectively. These results suggest that epidermal growth factor may be necessary for the normal course of pregnancy.

Anti-nerve growth factor antiserum antagonizes the masculinizing effect of testosterone in neonatal female rat brain

Anti-nerve growth factor antiserum antagonizes the masculinizing effect of testosterone in neonatal female rat brain

Neonatal treatment with nerve growth factor antiserum eliminates cholinergic sympathetic innervation of rat sweat glands

Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.