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Nerve growth factor (NGF) was recently found to be largely associated with sedimentable fractions of adult rat brain and treatments of the fractions by alkaline pH increased the measurable amount of their NGF antigen as well as its solubilization [M.C. Hoener, E. Hewitt, J.M. Conner, J.W. Costello and S. Varon, Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res., 728 (1996) 47-56; M.C. Hoener and S. Varon, Effects of sodium chloride, Triton X-100, and alkaline pH on the measurable contents and sedimentability of the nerve growth factor (NGF) antigen in adult rat hippocampal tissue extracts, J. Neurosci. Res., in press (1997); C. Zettler, D.C.McL. Bridges, X.-F. Zhou and R.A. Rush, Detection of increased tissue concentrations of nerve growth factor with improved extraction procedure, J. Neurosci. Res., 46 (1996) 581-594]. We have further investigated the reversibility of these pH effects. Reversal of the pH of an adult rat hippocampal tissue extract from 10.5 to 7.4 led to an almost complete transfer of NGF back from nonsedimentable to sedimentable fractions and to a remasking of the previously unmasked portion of NGF antigen. Thus, molecules causing masking and sedimentation of NGF at pH 7.4 were likely to be present in the alkaline extract. A gel filtration column in PBS, pH 10.5 was used to separate such putative binding molecules from the NGF. All of the NGF antigen from rat hippocampal alkaline extract was found to elute with 19 kDa fractions. The same apparent molecular weight was found for mouse submaxillary beta-NGF and recombinant human beta-NGF. Masking and sedimentation no longer occurred when newly generated 19 kDa rat brain NGF was returned to pH 7.4. When high molecular weight fractions derived from the same gel filtration (in PBS, pH 10.5) were added back to the 19 kDa NGF pool at pH 7.4 and the mixture incubated and centrifuged, the measurability of 19 kDa rat brain NGF antigen was markedly reduced and half of the antigen was recovered in sedimentable fractions. Similar but less dramatic results were obtained when mixing the same high molecular weight fractions with 19 kDa mouse or human beta-NGF. These findings provide new opportunities to identify molecules to which NGF may be bound within intact brain tissues.

Nerve growth factor (NGF) was found to be largely associated with sedimentable fractions of hippocampal and other neural tissues of the adult rat (Hoener et al.: Brain Res 728:47–56, 1996), verified by both bioassay and ELISA techniques. In the present study, the ELISA assay conditions were improved and simplified. Bovine serum albumin was needed in the phosphate buffered saline for maximal measurability of NGF antigen. Hippocampal tissue sonicates were separated into nonsedimentable supernatant and sedimentable pellet fractions. Individual or combined treatments with sodium chloride, Triton X-100, and pH were applied to the samples for possible effects on the i) measurable content of NGF antigen and ii) distribution of sedimentable and nonsedimentable forms. The amount of measurable NGF antigen was found to be increased in a dose dependent fashion by sodium chloride between 0.15 and 0.35 M, Triton X-100 between 0 and 0.5%, and pH between 8.5 and 10.5. The same treatment that led to maximal measurable NGF levels (0.7% Triton X-100 and pH 10.5) also caused the release of the NGF antigen from sedimentable to nonsedimentable fractions. Similar findings regarding maximal NGF antigen levels and release were seen for treatments applied to the sonicate before separation into a supernatant and pellet fraction. J. Neurosci. Res. 49:508–514, 1997. © 1997 Wiley-Liss, Inc.

Previous studies revealed that NGF-like immunoreactivity is present in cells from the adult rat anterior pituitary lobe, both in vivo and in vitro, and that in both situations NGF colocalizes with the thyroid-stimulating hormone (TSH). More recently, brain-derived neurotrophic factor (BDNF) was similarly found to occur in the anterior pituitary tissue, again with a general colocalization with TSH. In the present study, we have extended the use of adult rat anterior pituitary cultures to show their content of BDNF-immunoreactive cells and their main colocalization with TSH. We have also explored the question of whether neurotrophins nerve growth factor (NGF) and/or BDNF are actually produced within anterior pituitary cells. Use of the sensitive method reverse transcription-polymerase chain reaction (RT-PCR) has allowed us to confirm the presence of NGF and BDNF mRNAs in the cell suspension freshly derived from adult anterior pituitary. In situ hybridization techniques applied to the cell cultures from such a suspension, however, have revealed only a variable presence of NGF mRNA-positive cells but no recognizable BDNF mRNA. Thus, the question of whether the two neurotrophins are produced within the very cells whose immunoreactive content can be recognized remains an open one.

We have used enriched dissociated, low density cultures of neonatal rat corticospinal motor neurons to evaluate the survival-promoting effect of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) and the ciliary neurotrophic factor (CNTF). Our current findings demonstrated that CNTF promoted the survival of corticospinal motor neurons, in the same fashion and at an equivalent potency, as was previously described using a different assay system. Among the other factors tested, we also found that NT-4 and GDNF increased the number of surviving neurons in a dose-dependent manner, whereas NGF, BDNF and NT-3 showed no survival promoting effect on corticospinal motor neurons.

A sensitive immunohistochemical technique was used, along with highly specific affinity-purified antibodies to brain-derived neurotrophic factor (BDNF), to generate a detailed mapping of BDNF immunoreactivity (BDNF-ir) throughout the adult rat CNS. A parallel analysis of sites of BDNF synthesis was performed within situhybridization techniques using a cRNA probe to the exon encoding mature rat BDNF protein. These combined data revealed (1) groups of cell bodies containing diffuse BDNF-ir throughout the CNS that were strongly correlated with fields of cells containing BDNF mRNA; (2) varying degrees of BDNF-ir outside of cell bodies, in what appeared to be fibers and/or terminals; and (3) many regions containing extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord). The latter observation suggested that in these regions BDNF was derived from anterograde axonal transport by afferent systems. In the two cases in which this hypothesis was tested by the elimination of select afferents, BDNF immunostaining was completely eliminated. These data, along with the observation that BDNF-ir was rarely found within dendrites or fibersen passage, suggest that BDNF protein produced in adult CNS neurons is polarized primarily along axonal processes and is preferentially stored in terminals within the innervation target.

Previous studies in adult rats have demonstrated that the neurotrophin, brain-derived neurotrophic factor (BDNF), is present in virtually all cells of the pituitary intermediate lobe. In the present study, we demonstrate that cells cultured from adult intermediate lobe pituitary (ILP) rapidly lose their BDNF immunoreactivity (IR). Furthermore, a similar loss of immunostaining occurs in whole (undissociated) ILP within 30 min after removal from the rat. However, when the dopamine agonist apomorphine is present throughout the dissociation procedure and during cultivation, BDNF-IR is preserved. Supplying apomorphine only during either dissociation or cultivation did not prevent the loss of BDNF-IR in the 24 h cultures. These results suggest that a tonic dopaminergic stimulus is required to maintain BDNF-IR in ILP cells.

Immunohistochemical techniques were used to determine the distribution of brain-derived neurotrophic factor (BDNF) in the adult rat pituitary gland. In the posterior lobe, BDNF staining was found only within fibers. Within the intermediate lobe, nearly all cells were labeled intensely for BDNF. In the anterior lobe, varying degrees of BDNF immunoreactivity were observed exclusively in cells shown by double-labeling techniques to contain thyroid stimulating hormone (TSH), although not all TSH-positive cells contained detectable BDNF labeling. These results are consistent with and extend information from previous studies demonstrating BDNF expression in the pituitary gland and further support the idea that this neurotrophin plays a role in endocrine function.

Initial studies had revealed that the bioactivity of nerve growth factor (NGF) in sonicates of adult rat hippocampal formation (HF) is several-fold greater in their pellet than their supernatant fractions. Such observations have prompted an analysis of NGF antigen (NGF-Ag) contents in pellets and supernatants from a variety of adult rat CNS tissues, both in the absence and the presence of exogenous β-NGF. With HF tissues, NGF-Ag in the supernatants was comparable to most literature values, but pellet NGF-Ag was 3 to 5 times that amount. All other CNS tissue sonicates also revealed 3–6 fold higher NGF-Ag in their pellets than their supernatants, hence overall NGF-Ag contents were greatly in excess of reported ones. Presentation of mouse β-NGF to a tissue, its sonicate, or its standard pellet resulted in a transfer to the final pellet of 30–50% of the added soluble NGF-Ag (and 30% of the added bioactivity). This percentage is much lower than that present in native pellets (80%), suggesting that the association of endogenous NGF with particulate matter may involve at least two compartments. Treatments of pellets with salt, alkaline pH, and/or the detergent Triton X-100 have revealed a third subset, namely additional pellet NGF-Ag that was not initially recognized by the antibody in our ELISA assay. The treatments also caused substantial release of NGF from pellet to supernatant. Further studies should clarify the nature of the association between NGF and the three subsets of pellet NGF and allow the investigation of the pellet molecules responsible for it.

The neurotrophin family of growth factors consists of proteins sharing a sizable degree of amino acid sequence and structural homology. These similarities greatly increase the probability that antibodies directed against any single neurotrophin may cross-react with other family-members. Various investigators have documented such cross-reactivity can occur under experimental conditions, although the extent of cross-reactivity reported in different studies has varied greatly. Although the use of different antibody preparations may have contributed to the differing degrees of cross-reactivity observed, it is important to note that different assay conditions were also used to evaluate cross-reactivity in the various studies. Little information is currently known about how antibody-antigen interactions vary as a function of the assay conditions used for the evaluation. The present study addressed this question by evaluating the cross-reactivity occurring between various neurotrophins and a single preparation of antibodies directed against purified mouse nerve growth factor-beta (beta-NGF) in a wide variety of immunological assay systems. Our results indicate that cross-reactivity between NGF antibodies and related neurotrophins can vary greatly depending upon the assay system chosen for the evaluation. These data strongly argue against transferring cross-reactivity data across various assay systems.

In the present study, the authors reevaluated the temporal course and properties of neurotrophic activities present in the fluid accumulating in the silicone-chamber model for nerve regeneration. The fluid collected from silicone chambers was tested in four different dissociated neuronal cell cultures. Furthermore, the activity of the chamber fluid was examined, using a cell blot technique. There was one major peak of neurotrophic activity and this activity peaked early, about 3 to 6 hr after nerve injury. Results also indicate that the chamber fluid contains at least two types of neurotrophic activities, namely nerve growth factor and ciliary neurotrophic factor.

Following cholinergic denervation of the hippocampal formation, peripheral sympathetic fibers from the superior cervical ganglion (SCG) sprout into hippocampal tissue. The molecular mechanism controlling this process is unknown, although a role by trophic factors seems likely. In the present study, neonatal SCG were used as biological probes to investigate the association between NGF-immunoreactive regions in the hippocampal formation and areas innervated by regrowing sympathetic fibers. Cholinergic deafferentation of the hippocampal formation was achieved by a fimbriafornix transection and neonatal SCG were placed into the lesion cavity, abutting the rostral pole of the hippocampus. At 16 days following ganglia transplantation, NGF immunoreactivity within the hippocampal formation appeared indistinguishable from unlesioned controls and was localized within the mossy fiber region of the dentate gyrus and CA3 and CA2 hippocampal subfields. Sympathetic innervation, revealed in adjacent sections by dopamine β-hydroxylase or p75LNGFR immunoreactivity, was also restricted primarily to the mossy fiber region. Ablation of the entorhinal cortex at the time of transplantation resulted in the appearance of an additional discrete band of NGF immunoreactivity within the outer molecular layer of the dentate gyrus. In animals receiving an entorhinal lesion concurrent with the SCG transplant, sympathetic fibers were observed not only within the mossy fiber region but also within the outer molecular layer of the dentate gyrus where the lesion-induced NGF immunoreactivity appeared. These results support the hypothesis that topographic distributions of NGF may be used in the hippocampal formation to define terminal fields for sprouting NGF-sensitive neuronal populations.

The distribution of nerve growth factor (NGF), the prototypic neurotropin, within the basal forebrain and hippocampal formation of young adult monkeys and aged humans was characterized with an affinity purified polyclonal beta-NGF antibody raised against mouse beta-NGF. In the basal forebrain of both primates, a granular NGF-like immunoreactive (ir) reaction product was observed within neurons of the medial septum, nucleus of the diagonal band, and nucleus basalis of Meynert. NGF-like immunoreactivity exclusively colocalized within p75 NGF receptor (NGFR) containing basal forebrain neurons. The intensity of NGF immunolabeling varied between cell bodies. Many NGF-ir perikarya were highly immunoreactive. In other basal forebrain neurons, NGF-like immunoreactivity was either undetectable or minimally expressed. In the hippocampus of both species, NGF-like immunoreactivity was mainly localized within the hilus of the dentate gyrus and within CA3 and CA2 hippocampal subfields. A marked diminution in NGF-like staining was seen in CA1. Within the hippocampal formation, NGF-like immunoreactivity was heaviest within the neuropil of stratum radiatum, intermediate in stratum oriens, and lightest in stratum pyramidal. NGF-like immunoreactivity was not found within the granule or pyramidal cells of the dentate gyrus and hippocampal formation, respectively. These findings demonstrate the presence of an NGF-like antigen in association with monkey and human magnocellular basal forebrain neurons and within their hippocampal target sites. This lends support to the hypothesis that NGF is internalized from sources located within target regions of the primate cholinergic basal forebrain neurons and is retrogradely transported to these cell bodies where the NGF trophic effect likely occurs.

The development and maturation of cells depends not only on their genetic history, but also on sequences and combinations of environmental signals appropriate to their developmental age. Early postnatal rat sympathetic neurons are dependent on nerve growth factor (NGF) for survival in vivo and in vitro , but earlier sympathetic neuroblasts may not require NGF. Ciliary neurotrophic factor (CNTF) provides short-term in vitro trophic support to embryonic and neonatal sympathetic neurons, but its role in vivo is not understood. In this study we examined further the capability of CNTF to support neonatal rat superior cervical ganglion (SCG) sympathetic neurons in vitro and the effect of CNTF on the trophic activities of NGF. SCG neurons cultured with either CNTF or NGF survived for 24 hr in low-density cultures depleted of nonneuronal cells, in contrast to neurons with neither factor. However, with CNTF only a fraction of the NGF-maintained number of neurons survived for 6 days. CNTF given in combination with NGF in these nonneuron-depleted cultures produced a significant decrease in the number of neurons surviving for 6 days, compared to the number supported by NGF alone. If such cultures were supplemented with Schwann cells, very different results were obtained: CNTF alone supported the 6-day survival of 80% as many neurons as did NGF, and the combination of CNTF with NGF produced no decrease in neuronal survival. Antibody to NGF did not block the support provided by CNTF and Schwann cells. Immunostaining for the low-affinity NGF receptor (LNGFR), intense in the NGF-supported neurons, was absent in the CNTF-supported neurons and reduced in the neurons exposed to the combination of NGF and CNTF. These results show that CNTF can act synergistically with a Schwann cell-derived agent to provide trophic support to neonatal sympathetic neurons, and that it can down-regulate the responsiveness of those neurons to NGF.

Neurotrophic factors promote maintenance, repair and regeneration of selected neurons in vitro and in vivo. They include nerve growth factor (NGF) and other neurotrophins, ciliary neurotrophic factor (CNTF) and other cytokines, and a number of growth factors. Molecular properties of the trophic proteins and their transducing receptors are increasingly well characterized. Interest in the neurotrophic factors has greatly accelerated over the past decade with accumulating evidence that they play important roles in the adult mammalian CNS as well as in its development. Adult in vivo models for degenerative CNS conditions have demonstrated the ability of exogenous neurotrophic factors (NTFs) to protect against neuronal damage induced by traumatic or chemical experimental lesions, particularly with regard to easily identifiable neurons such as cholinergic and dopaminergic ones. Other models have shown beneficial effects of NTF administration with regard to axonal regeneration inside CNS tissue.

Ramified parenchymal microglia may provide immune surveillance in the nervous system and become activated in response to injury, showing increases in antigens found on macrophages, e.g. CD4 and MHCs. We investigated in adult rats the effects of a 2-week intraventricular infusion with ciliary neurotrophic factor (CNTF), a nervous system-associated cytokine, on microglia of the normal and injured corpus callosum. CNTF caused morphological changes, induced the expression of low-affinity nerve growth factor receptor and CD4 and increased the expression of complement receptor 3. Such changes were also observed after treatment of pure cultures of neonatal rat microglial cells with highly purified CNTF, suggesting a direct responsiveness to CNTF. Thus, endogenous astroglial and Schwann cell-derived CNTF may be an important component of the immune responses of the nervous system.

Corticospinal neurons were identified in cell cultures of neonatal rat cortex by immunostaining of cholera toxin B subunit (CTB), retrogradely transported from the cervical part of the spinal cord. The CTB-immunoreactive neurons were larger than the neurons in the overall (unstained) neuronal population and represented a small fraction of it (average of 0.3%) after 6 hours in vitro. The number of both total and CTB-labeled neurons declined progressively with time in culture. The neuronal death was, however, markedly faster in the CTB-labeled neuronal population than in the overall neuronal population. Ciliary neurotrophic factor (CNTF) promoted the survival of CTB-positive corticospinal neurons in a dose-dependent manner; with CNTF, the death rate of the CTB-labeled neurons became identical to that of the overall population.

Nerve growth factor (NGF) has long been recognized for its neurotrophic actions upon sensory and sympathetic neurons of the PNS and upon cholinergic neurons of the CNS basal forebrain. Much evidence, however, suggests that potential reciprocal interactions between NGF and various endocrine systems may also occur. In the present investigation we used immunohistochemical techniques to investigate whether NGF is found within the adult rat pituitary, thereby suggesting a possible interaction within this critical neuroendocrine gland. Our results indicate that NGF immunoreactivity is present only within the anterior lobe of the male and female rat pituitary and is exclusively localized within cells immunoreactive for thyroid stimulating hormone (TSH).

Nerve growth factor (NGF) can induce sprouting of axotomized adult rat medial septum cholinergic neurons and promote their regeneration into septohippocampal nerve grafts and hippocampal formation. This study investigated the potential neurotropic (chemotactic/attracting) action of NGF in the adult rat cholinergic septohippocampal regeneration model. (i) Some animals received sciatic nerve grafts between the disconnected septum and hippocampal formations on each side. A 4-week infusion with NGF into the rostral portion of the lateral ventricle induced sprouting of cholinergic fibers in the dorsolateral septum with a gradient toward the lateral ventricle. However, the number of cholinergic axons entering the nerve bridge was only one-third that observed in vehicle-infused animals, suggesting that NGF had diverted many of the regrowing axons away from the nerve toward the ventricle. (ii) In animals implanted with nerves for 2 weeks and concurrently infused with NGF into the fornix, proximal to the lesion and grafts, cholinergic sprouting occurred in the mediodorsal septum, i.e., was oriented toward the infused fornix. Essentially no fibers had entered the nerve bridge, suggesting that all regrowing fibers had remained near the NGF source. (iii) When animals with a unilateral fimbria-fornix transection (but no nerve graft) were infused with NGF into the lateral ventricle on the opposite side, cholinergic sprouting was oriented toward the midline of the septum. (iv) Infusion of low doses of NGF directly into the (lesioned) septum induced a sprouting response localized around the infusion site. (v) No sprouting occurred when intraventricular NGF infusion was applied to normal (nonlesioned) animals. These results show that the location of an NGF source can control the orientation of the outgrowing cholinergic axons, and indicate that NGF has a neurotropic action in the adult animal in vivo, as it has been shown to have for developing neurons both in vitro and in vivo.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septa) or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.

Cells in the dissociated state from the sympathetic ganglia (SG) of 11-day-old chick embryos, and monolayer cultures of these cells are used to illustrate some of th extrinsic influences that regulate neuronal performance. In culture, the survival of SG neurons can be measured, as an assay for survival-promoting agents. Among the requirements of the SG discussed are: (1) nerve growth factor and other trophic factors that can replace it, (2) serum, and a defined mixture (N1) that can substitute for it, and (3) a minimal presence of non-neuronal cells. Also reviewed are factors that confer neurite-promoting competence on certain culture substrata. Suspensions of SG cells permit analysis of "short-latency" events triggered within minutes of the presentation of nerve growth factors and provide an insight into its possible mode of action. The most striking such event is its control over Na+/K+ pumps, since ionic control is a fundamental feature of living cells and may well mediate their regulation by trophic factors, hormones or mitogens.

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen-stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.

The well-documented but little-understood failure of lengthy axonal regeneration after injury of the adult mammalian CNS may be caused by an insufficient availability of local growth-promoting factors. If so, identifying and supplying the missing factors may result in better central axonal regeneration. This hypothesis was tested in an adult rat CNS model in which peripheral nerve grafts were placed into a lesion cavity between the septum and hippocampal formation. Continuous infusion of nerve growth factor (NGF) into the dorsal hippocampal tissue dramatically enhanced and accelerated the regrowth and penetration of cholinergic axons into the hippocampal formation. Thus, NGF can overcome the apparent resistance of the hippocampal CNS tissue to cholinergic reinnervation.

Previous studies have demonstrated that partial cortical devascularization results in increased levels of nerve growth factor protein within the tissue immediately surrounding the infarcted region. In the present study, we have used this lesion model to further characterize the nerve growth factor increase by investigating: (i) the time course for this phenomenon; (ii) the impact of the devascularizing lesion on cortical regions not directly impinged upon by the lesion; and (iii) the response of nerve growth factor-sensitive nucleus basalis neurons providing afferent cortical innervation to the increased availability of nerve growth factor within their target territory. Our results indicate that, within the infarcted cortex, nerve growth factor levels increase rapidly following the lesion (up 51% by one day post lesion), reach a maximum of 136% above controls by three days and undergo a slow decline back to baseline levels by 23 days. Within the frontal and cingulate cortices, which are not devascularized by the lesion and show no signs of pathological damage, nerve growth factor levels increase over a similar time course, and with a similar magnitude, to those in the lesioned cortex. Nerve growth factor-sensitive nucleus basalis neurons on the side ipsilateral to the lesion respond to increased cortical nerve growth factor levels with an increased accumulation of nerve growth factor within their cell bodies (revealed by nerve growth factor immunohistochemistry and quantitative enzyme-linked immunosorbent assay) which was apparent at three days following the lesion, but no longer discernible at seven or 14 days or later. The present study investigated a model of cortical devascularization for its ability to alter nerve growth factor levels within the cortex. Nerve growth factor levels were rapidly increased within the infarcted cortical tissue beneath the lesion but were also elevated to a similar extent, and with a similar time course, in cortical regions not directly impinged upon by the lesion. The retrograde impact of elevated cortical nerve growth factor levels was demonstrated by an increased accumulation of nerve growth factor within the cell bodies of nucleus basalis neurons providing innervation to the cortex. This lesion model should provide a potential avenue for investigating the functional role(s) of nerve growth factor in the intact and lesioned adult central nervous system, as well as the internal mechanisms for regulating its synthesis, release, uptake, and degradation.

In the current investigation, we have examined the developmental profile of nerve growth factor immunoreactivity (NGF-ir) in the postnatal rat. During the first 3 weeks after birth, NGF-ir was observed within the hippocampal mossy fiber region, where it persists throughout adulthood and appeared transiently within three additional zones – the dentate gyrus supragranular zone, the tenia tecta/intermediate lateral septum, and the cingulate/retrosplenial cortex. In all cases, the appearance of NGF-ir progressed in a rostrocaudal pattern over time. A strong correlation was seen between the pattern of NGF-ir and cholinergic innervation in the dentate gyrus supragranular zone, both spatially and temporally, suggesting that NGF may direct the innervation of cholinergic afferents to this region. A spatial correlation was also observed between NGF-ir and cholinergic innervation within the retrosplenial cortex and tenia tecta. With our current techniques, however, we were unable to determine at what point during development the adult-like pattern of cholinergic terminal innervation in these regions occurred and, thus, were not able establish a temporal correlation in these regions. Within the cingulate cortex, there was no evidence suggesting that the developmental appearance of NGF-ir in this region was associated with a specific enhancement of cholinergic innervation. Thus, the results of the current investigation clearly identify the presence of transiently occurring zones of NGF-ir during postnatal CNS development, although defining their exact functional role will require additional investigation.

Nerve growth factor (NGF) has been postulated to play an important role in the process of sympathetic sprouting into the septally deafferented hippocampal formation. In the current investigation we have used transplants of NGF-dependent neonatal superior cervical ganglion (SCG) neurons to investigate the influence of NGF and septal denervation (either alone or in combination with one another) upon neuronal survival and intrahippocampal sprouting. In the current model, SCG transplants were placed onto the dorsal surface of the CA1 hippocampal subfield and a continuous infusion device was used to deliver either NGF or vehicle into the hippocampal parenchyma. Following 15 days of vehicle infusion, little or no sympathetic neuronal survival was observed and no hippocampal fiber outgrowth was detected. NGF infusions, however, promoted both neuronal survival and intrahippocampal fiber outgrowth directed mainly toward the location of the infusion cannula. Septal denervation, achieved by either a septal ablation or fimbria-fornix transection, had no discernible impact upon SCG neuronal survival or fiber outgrowth, with or without NGF infusions. Similar results were also obtained when SCG were transplanted directly within the cortex, with or without an intracortical infusion. It appears, therefore, that NGF may be a sufficient, as well as necessary, component for eliciting and guiding the invasion of a tissue by NGF-sensitive fibers.

Little is known about the neurotrophic factors that may regulate maintenance, growth, and/or repair of corticospinal motor neurons (CSMN) in the developing or the adult mammal. We have developed an adult rat in vivo model of CSMN injury involving (i) bilateral prelabeling of CSMN with a cervical spinal cord injection of cholera toxin B subunit (CTB), (ii) unilateral axotomy close to the cell bodies by an intracortical lesion between cell layer V and the corpus callosum, and (iii) implantation of a continuous infusion device in the cortical parenchyma near the lesion. Two weeks later, coronal sections of the cortex are immunostained for CTB, and CTB-stained neurons are counted over defined section areas and compared to those on the contralateral (nonlesioned, noninfused) side. CTB-stainable neurons were 30-40% of the control side when the lesion was about 200 microns from the deeper face of the cell layer, and survival increased with increasing lesion depths. The model can be used to assess protective effects of potential CSMN trophic factors. The low survival achieved with the more superficial lesions (200 and 300 microns) was markedly improved by continuous infusion of ciliary neurotrophic factor at 0.1 to 1.0 microgram/day.

The effect of intraspinally implanted predegenerated peripheral nerve grafts and/or conditioning lesions on the regenerative capacity of central ascending sensory axons was investigated in the adult rat. Regenerating sensory fibers were analyzed after their transganglionic labeling with cholera toxin B subunit, usually 1 month after implantation. A conditioning lesion (transection of the tibial and peroneal nerve) caused a fivefold increase in the number of sensory fibers within the fresh graft when applied on the day of grafting and a sevenfold increase when applied 1 week before. In the latter case, a small portion of the fibers (10%) had reached the rostral end of the nerve graft. In the absence of a conditioning lesion, the number of fibers regenerating into a predegenerated nerve graft (collected from the distal part of the peroneal nerve that had been axotomized 1 week earlier) was similar to that found in a fresh graft. However, predegenerated grafts received three and five times more fibers than a fresh graft when conditioning lesions were applied on or 1 week before the day of grafting. With the combination of a predegenerated graft and a 1-week conditioning, most (> 90%) of the regenerating fibers had reached the rostral graft-host border. In animals with a fresh graft, a portion of the axotomized fibers formed terminal club-like structures. Much fewer fibers displayed such clubs in animals with a predegenerated graft or a conditioning lesion, suggesting a preventive action of either treatment. A time-course study with the combined treatments showed that regenerating sensory fibers had already entered the graft after 3 days. Between 1 and 2 weeks, a maximum number of fibers had reached the rostral end of the nerve graft. However, after 2 months, the number of fibers was decreased, i.e., the initial advantage of predegeneration had diminished. The current results demonstrate the necessity of a conditioning lesion for successful regeneration of central sensory fibers, possibly resulting from an earlier induction of the neuronal growth response which allows the axon to enter the graft before the formation of a graft-host barrier. The predegeneration of the nerve graft augments the growth response of the axotomized central sensory fibers, probably by providing a more supportive terrain and/or enhancement of the neuronal response. The presence of a large number of fibers at the rostral graft-host border now provides the opportunity to investigate the effects of neurotrophic factors on the regenerative capacity of the ascending rat sensory fibers into the denervated spinal cord in vivo.

The hypothesis that neurotrophic factors play important roles in the adult central nervous system (CNS) has been successfully investigated in the past decade with regard to experimental and pathologic situations. Trophic roles in adult CNS axonal regeneration, on the other hand, have received much less attention. We review three groups of recent studies that demonstrate the relevance of nerve growth factor (NGF) for the regeneration of selected axons into adult central nervous tissue. The first group concerns a septohippocampal model where transected septal cholinergic axons are allowed to regrow into the hippocampal formation through a peripheral nerve bridge implanted into the transection lesion gap. NGF is required in the bridge, enhances penetration of the hippocampal tissue when infused there, and both attracts and promotes sprouting within the septum when infused in the lateral ventricle or the septal tissue itself. The second group of studies concerns the development of a spinal cord sensory regeneration model, where dorsal root ganglionic axons regrow into a nerve bridge placed within the dorsal spinal cord. Preliminary data indicate that NGF infusion rostral to the bridge once again promotes substantial penetration of the adult cord tissue by the regenerating NGF-sensitive fibers. In the third group of studies, attention has been shifted to the location of endogenous NGF in the adult rat hippocampal formation and the normal or lesion-induced occurrence of extrasomal NGF immunoreactivity. These regions of anchored NGF have the ability to attract NGF-sensitive growing axons and may provide opportunities to investigate local cues for final definition of terminal fields.

To assess the possible role of trophic factors in lesion-induced plasticity, we have used a sensitive immunohistochemical technique to evaluate changes in nerve growth factor (NGF) staining in the hippocampal formation 3, 8, 16, and 30 days following entorhinal cortex lesions. Our results indicate that a band of NGF immunoreactivity appears in the outer molecular layer of the ipsilateral dentate gyrus following entorhinal ablation. The distribution of the NGF-immunoreactive band exactly coincides with the distribution of sprouting cholinergic terminals revealed by acetylcholinesterase histochemistry or NGF-receptor immunostaining. Increased NGF-immunoreactivity is detectable at 3 days postlesion, is most intense at 8 days, and decreases to near control levels by 30 days. Lesion-induced increases in NGF immunostaining also occur in animals in which septohippocampal fibers had been removed by prior destruction of the fimbria-fornix. Increases in NGF-immunoreactivity, however, are substantially reduced in animals receiving intraventricular injections of colchicine, which presumably blocks NGF release. These results indicate that (1) increases in NGF immunostaining, which occur following entorhinal lesions, precede any changes in cholinergic sprouting parameters and are greatest during the period of maximal cholinergic sprouting; (2) increased NGF-immunoreactivity is not due to NGF binding by septohippocampal fibers; and (3) increased NGF-immunoreactivity appears to depend on the release of NGF by neurons that produce it. We hypothesize that, following entorhinal lesions, NGF immunostaining within the hippocampal formation may represent NGF “anchored” within the tissue and that NGF accumulation by such a mechanism may direct the sprouting response of NGF-sensitive cholinergic neurons. © 1994 Wiley-Liss, Inc.

We examined the effects of changes caused by the blocking of protein and RNA synthesis on neurite outgrowth from neurons of the central nervous system (CNS) in primary culture. Exposure to cycloheximide and actinomycin-D led to dramatic increases in the length of neurites in cultures of neurons from various rat or chick CNS regions. Inhibitor-induced neurite outgrowth was observed (1) from dopaminergic neurons in mixed cultures of the rat substantia nigra or (2) in pure cultures of rat and chick neurons grown on a polyornithine/laminin substratum. These results suggest that neurite outgrowth from CNS neurons is kept restricted, at least in culture, by the continuous production of a labile neurite-inhibiting protein intrinsic to the neurons, which rapidly decays following inhibition of protein or RNA synthesis. 1994 John Wiley & Sons, Inc.

We have investigated the neuroprotective effects of recombinant human ciliary neurotrophic factor (CNTF) for injured dopaminergic neurons of the adult rat substantia nigra compacta. Fourteen days after a unilateral transection of the nigrostriatal pathway two-thirds of the neurons (identified by retrograde labeling) had degenerated. In sharp contrast, 73% (a few cases, > 90%) of this cell loss was prevented by continuous infusion of CNTF close to the injured neurons. However, CNTF did not prevent the disappearance of the transmitter-synthesizing enzyme tyrosine hydroxylase. Thus, CNTF has potent neurotrophic effects for injured adult rat dopaminergic substantia nigra neurons, whose degeneration plays a major causative role in Parkinson disease.

A proportion of developing oligodendrocytes undergo natural cell death by apoptosis, and mature oligodendrocytes die, either by apoptosis or necrosis, in response to injurious signals such as cytotoxic cytokines and complement. Ciliary neurotrophic factor (CNTF), a trophic factor found in astrocytes in the central nervous system (CNS), promoted the survival and maturation of cultured oligodendrocytes. This trophic factor also protected oligodendrocytes from death induced by tumor necrosis factors (apoptosis) but not against complement (necrosis). These results suggest that CNTF functions in the survival of oligodendrocytes during development and may lead to therapeutic approaches for degenerative diseases of the CNS that involve oligodendrocyte destruction.

Ciliary neurotrophic factor (CNTF) was found to promote the expression of tyrosine hydroxylase (TH) immunoreactivity by cultured noradrenergic neurons from the locus coeruleus (LC) of E18 rat fetuses, but only in the concomitant presence of norepinephrine (NE), their own neurotransmitter. The number of TH-positive cells in LC cultures was shown to decrease by 65% within 3 days and by 75% after 6 days. Treatment with 10 TU/ml human recombinant CNTF together with 1 microM NE was able to fully maintain the initial number of TH-positive neurons for 3 days. This effect, however, was no longer seen after 6 days of continuous exposure. A 24-hr treatment with CNTF/NE was capable of completely restoring the initial number of TH-positive cells, even if its addition was delayed for 2 days. Moreover, when its addition was delayed for 5 days, CNTF/NE restored approximately 80% of the TH-positive neurons that were initially present. These results suggest that the disappearance of TH-positive neurons in LC cultures is not due to their death, but rather to the reduced expression of TH and that the simultaneous exposure to CNTF and NE upregulates TH. Effects on TH-positive cell number were not evoked by CNTF or NE alone. The CNTF/NE effect was dependent on protein synthesis, but was only partially inhibited by RNA synthesis inhibitors, suggesting that both transcription from preexisting mRNA and synthesis of new RNA were stimulated. The effect of CNTF/NE was mediated by alpha 2-adrenoceptors, since it was blocked by alpha 2-antagonists and since alpha 2-agonists were able to substitute for NE. Our results suggest a novel mechanism of regulation of the phenotype of the noradrenergic LC neuron, involving the collaborative influences of CNTF and norepinephrine, their own neurotransmitter.

Developing spinal motor neurons (SMN) express low-affinity nerve growth factor receptors (LNGFR) but not high-affinity transducing NGF receptors. Moreover, SMN are not supported by NGF in vitro. In the normal adult rat most SMN are not LNGFR immunoreactive (LNGFR-IR), but they transiently reexpress LNGFR (though not the high-affinity receptor) after peripheral nerve injury. With a cut lesion of the sciatic nerve (when only a neuroma forms), the number of LNGFR-IR SMN at L4-L6 rapidly increases to a maximum between day 1 and 7 and returns to baseline levels by day 30. After a crush lesion (accompanied by regeneration to the muscle), LNGFR-IR SMN appear in about the same numbers, but they start to disappear 1 week later. We speculate that the similar appearance and differential decline of LNGFR-IR seen after the two types of lesions are regulated by the availability of a common signal such as ciliary neurotrophic factor. The adult SMN model provides a good opportunity to investigate the reexpression of LNGFR after peripheral nerve injury, and more generally, the unknown role and regulation of LNGFR.

Using immunohistochemical techniques, we have previously localized nerve growth factor (NGF)-like immunoreactivity in the normal adult rat centralnervous system (CNS) exclusively in the hippocampal mossy fiber region andwithin basal forebrain cholinergic neurons—a cell population believed to be primary NGF consumers within the CNS. In the present investigation, we have attempted to identify potential producers of NGF by pretreating animals with colchicine. Such a treatment would be expected to block microtubule-assisted neuritic transport mechanisms, thus preventing the accumulation of antigens normally obtained by retrograde transport and forcingthe accumulation of cell products normally exported anterogradely. Forty-eight hours after colchicine administration within their innervation territories, basal forebrain cholinergic neurons showed a marked loss of NGF-like immunoreactivity. Conversely, following colchicine treatment, many new populations of NGF-like immunoreactive cells were detected, several of which have been previously observed with in situ hybridization techniques for NGF mRNA. Many NGF-like immunoreactive populations, however, were not previously recognized by in situ hybridization methods, including cells of the striatum, reticular thalamic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, lateral and medial septum, substantia innominata, and nucleus basalis. Furthermore, evidence is provided that colchicine-blocked, NGF-like immunoreactive neurons within the basal forebrain are not cholinergic, thus reinforcing the hypothesis that trophic support for these NGF-dependent neurons may be derived from distant and local sources. The distinctive distribution of NGF-like immunoreactive cells observed in this study strongly correlates with the reported distribution of NGF mRNA in CNS neurons, thus suggesting that our antibodies are uniquely recognizing NGF and not other related neurotrophins. © 1992 Wiley-Liss, Inc.

During development, oligodendrocytes are generated from a bipotential glial stem cell, the oligodendrocyte-type-2 astrocyte precursor (O-2A). O-2A cells are under the mitogenic influence of the platelet-derived growth factor (PDGF) released from type-1 astrocytes. In vitro experiments have shown that O-2A cells stimulated by PDGF are limited to a set number of divisions and then differentiate to oligodendrocytes by becoming unresponsive to the growth factor. In the healthy adult central nervous system, oligodendrocyte proliferation remains generally quiescent and is possibly under negative growth control. The view that O-2A lineage cells are capable of negatively regulating their own proliferation is supported by the demonstration that conditioned medium obtained from O-2A cultures inhibits their DNA synthesis. In addition to O-2A cells, the newly established CG4 cell line, a derivative of O-2A cells, was found to inhibit O-2A lineage cell proliferation. The antiproliferative activity was present in the media conditioned by CG4 cells that were expanded as undifferentiated O-2A precursors, as well as by CG4 cells induced to differentiate to nonproliferating oligodendrocytes. Moreover, the inhibitory activity was produced by CG4 cells (source cells) propagated by various mitogens. The inhibition of mitotic activity was nearly complete, dose-dependent, fully reversible, and exhibited when CG4 cells (test cells) were stimulated to divide by various mitogens, such as PDGF, basic fibroblast growth factor, or medium conditioned by the neuronal B104 cell line. The inhibition of proliferation was accompanied by the conversion of the phenotype of CG4 cells, from A2B5+/O4− precursors to A2B5−/O4+ prooligodendrocytes. This change of antigenic phenotype, like the inhibition of proliferation, was reversible. These results suggest that O-2A cells secrete an autocrine inhibitory factor capable of reversibly withdrawing them from the cell cycle, which suggests that the commitment to the oligodendrocyte lineage might proceed through more than one discrete stage. © 1992 Wiley-Liss, Inc.

We have studied the effects of ciliary neuronotrophic factor (CNTF) and nerve growth factor (NGF) on cultures of E14 rat spinal cord cells maintained for 7 days. The trophic factors were supplied at the day of seeding and every other day thereafter. Treatments with CNTF (human recombinant or purified from rat sciatic nerve, 100 TU/ml) resulted after 7 days in an increase, relative to control cultures, of: (i) the total number of neurons (identified by neurofilament protein and neuron-specific enolase immunostaining) that were not stained with choline, acetyltransferase (ChAT) and low affinity nerve growth factor receptor (LNGFR) antibodies; (ii) the number of motoneurons (0.5% of the neuronal population) as identified by size (greater than 25 microns), morphology and immunostaining for ChAT and LNGFR; and (iii) a population of small- to medium-sized (less than 25 microns), ChAT- and LNGFR-positive neurons, representing 5-10% of the total neuronal population. NGF treatments (mouse submaxillary beta NGF; 10-3000 TU/ml) were without effect on all 3 neuronal populations. Experiments in which CNTF administration was delayed revealed that the population of ChAT- and LNGFR-negative neurons and the population of motoneurons, were both dependent on CNTF for their survival. The third population, small ChAT and LNGFR-positive neurons, was not dependent on CNTF for survival but was induced by CNTF to express its two markers. These observations indicate that CNTF is a neuronotrophic factor for motoneurons, but that the effect of CNTF is not restricted to that cell population. In addition to its survival promoting effect, CNTF has also a regulatory role on the expression of ChAT and LNGFR for some spinal cord neurons.

This study describes the relationship between the concentration of intraventricularly infused nerve growth factor (NGF) and several responses by axotomized cholinergic medial septum neurons and normal cholinergic neostriatal neurons of the adult rat. NGF infused for 14 days starting either immediately after a unilateral fimbria-fornix transection or after a 2-week delay period elicited similar dose-response relationships for the maintenance or restoration of ChAT and NGF receptor positivity and cell body size and for intraseptal ‘sprouting’ of the axotomized medial septum neurons. Thus, in the medial septum it appears that the expression of ‘marker’ molecules, cell body size and the induction of ‘sprouting’ are regulated by virtually the same concentrations of NGF in the two treatment strategies. This suggests that NGF has a general regulatory role and injured but untreated neurons remain fully susceptible to NGF at least up to 2 weeks after the lesion. A 14-day infusion with NGF also induced an above-normal cell body size (hypertrophy) both in axotomized medial septum and in intact striatal cholinergic neurons. The hypertrophic response of normal striatal neurons required less NGF than did that of medial septum neurons. Since the striatal response began to be detectable at a similar concentration as that required for the full maintenance or restoration of ChAT and NGF receptor positivity it could be seen as an unwanted side-effect. The definition of a sub-optimal dose with which a significant, but not maximal response can be elicited will allow future evaluations of potentially additive or synergistic actions by other agents.

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified β-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Increasing recognition is given by neuroscientists to the fact that considerable plasticity is retained even in the adult mammalian central nervous system (CNS), i.e. that maintenance and function of adult CNS neurons continue to depend on and be regulated by agents presented to them by their humoral, extracellular matrix and cellular micro-environments. One major class of macro-molecular regulatory agents is that of special proteins controlling the performance of selected target neurons named neuronotrophic factors (NTFs), of which Nerve Growth Factor (NGF) is the first discovered and best characterized representative (Levi-Montalcini, 1987). NGF and other newly recognized NTFs have been traditionally investigated in the context of lesions in the peripheral nervous system (PNS) and in the course of their early development, both in vivo and in vitro. A recently articulated CNS neuronotrophic hypothesis (Varon et al., 1984) has proposed that i) adult CNS neurons also require support from their own NTFs, and ii) relative deficits of endogenous NTF may underlie neuronal damages in experimental and pathologic situations, and iii) exogenous NTF administration may reduce or prevent neuronal damage in such situations.

Using mild conditions of SDS-PAGE, i.e. no heating of the sample, and the PhastSystem (Pharmacia), we found that bFGF, either natural bovine or recombinant human migrated at a 27 kD position in addition to the classical 18 kD one. By the cell-blot technique, we found that the biological activity toward rat astroblasts and 3T3 mouse fibroblasts was always restricted to the 27 kD band. Partial heat denaturation experiments revealed a close correlation between the remaining biological activity of bFGF in solution and the ratio of the 27 kD band versus the 18 kD band seen on SDS gels. These observations suggest that the bFGF which is biologically active in solution migrates at an apparent Mr of 27 kD in our conditions of electrophoresis, keeping its biological activity after electrophoresis, and the molecules which are inactive (denatured) in solution migrate at 18 kD and remain inactive. These experimental conditions, in which the biological activity appears to be preserved, could be referred to as "non-denaturing SDS-polyacrylamide gel electrophoresis" and could be useful, associated to cell-blot, for the search and characterization of new growth factors active on cells in culture.

We report a quantitative method by which a single microculture can be examined for (i) cell morphology; (ii) cell number; (iii) DNA synthesis; and (iv) expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

The time course of degeneration of chick retinal ganglion cells was examined with Nissl stains and immunohistochemical methods for detection of substance P-like immunoreactive and nicotinic acetylcholine receptor immunoreactive neurons. Small lesions were made in the retinae, adjacent to the optic nerve head, and were subsequently sectioned parallel to the vitreal surface, permitting direct comparison of normal and axotomized retinal ganglion cells distal to the site of axon damage. At four and six days after surgery, a large number of degenerating cells with clear cytoplasm and pyknotic nuclei were seen. After eight, 10 and 14 days, many retinal ganglion cells displayed a chromatolytic response with dispersed Nissl granules, eccentric nuclei and the cells appeared crenulated. The number of apparently normal neurons in the ganglion cell layer in the axotomized region was reduced by about 50% six days following surgery, by about 70% on the 10th day and by about 75% on the 17th day. The remaining neurons in the ganglion cell layer were identified as displaced amacrine cells. From day 2 onwards, increased numbers of glial cells were present in the optic fibre, ganglion cell and inner plexiform layers. Many glial cells were enlarged and displayed extensive cytoplasmic processes, while others showed mitotic activity. Somata and proximal dendrites of retinal ganglion cells were intensely stained for substance P-like immunoreactivity at two and four days following surgery. At six, eight and 10 days, staining intensity was markedly reduced though still evident and at 14 and 17 days, substance P-like immunoreactivity had virtually disappeared. The persistence of limited substance P-like immuno-reactive ganglion cells 10 days after surgery indicates that these cells have a relatively protracted response to axotomy. Nicotinic acetylcholine receptor-like immunoreactivity in the ganglion cells at two and four days following axotomy was substantially reduced. The majority of faintly stained nicotinic acetylcholine receptor-like immunoreactive ganglion cells, as visualized in counterstained sections, did not exhibit pyknosis in the immediate period following axotomy. Double label studies demonstrated that substance P-like immunoreactive ganglion cells were distinct from the nicotinic acetylcholine receptor-like immuno-reactive ganglion cells. In a second set of experiments, nerve growth factor was then placed into the vitreous humor following intra-retinal axotomy. The somata, dendrites and proximal axons of lesioned substance P-like immunoreactive ganglion cells in these retinae were more intensely stained for a longer period of time and appeared more robust than cells from untreated retinae. Never growth factor had no discernible effect on either unlesioned substance P-like immunoreactive ganglion cells or on normal or axotomized nicotinic acetylcholine receptor-like immunoreactive ganglion cells. These results suggest that nerve growth factor may affect a selected subpopulation of axotomized retinal ganglion cells and enhance their ability to survive axotomy.

Survival in monolayer culture of 4-day (stage 23) chick embryo lumbar spinal cord neurons can be regulated by two opposing activities. One, spinal neuronotrophic activity, promotes neuronal survival; and the other, spinal neuronotoxic activity, eliminates the neurons from the culture even when the trophic support is present at an optimal concentration. Quantitative microbioassays for each activity are presented and used to measure the relative amounts of each agent within different sources including glial, muscle, and spinal cord cell-conditioned media and fluid collected from peripheral and central nervous tissue lesions. Although both activities were present in all of the sources tested, their concentrations in the wound fluids were orders of magnitude greater than in the conditioned media. The fluid-derived trophic activities were inactivated by heat and trypsin and nondialyzable, whereas all of the conditioned media-derived trophic activities were heat- and trypsin-resistant and dialyzable.

We have compared the anatomic and functional regeneration of a transected sciatic nerve following regrowth from its proximal stump through either preformed empty mesothelial chambers or autologous nerve grafts bridging a 10 mm gap. Within the mesothelial chambers an organized multifascicular nerve trunk forms between the proximal and distal stumps. After 3 months, distal segment cross sections from the mesothelial chamber and nerve graft groups did not differ with respect to axonal density or distribution of axonal diameters. Mean conduction velocities across the gaps were also similar, although the nerve graft group had a wider distribution of velocities. Little or no regeneration was evident when the gap between the nerve stumps was left empty. These results suggest that if the regrowing proximal stump is in an appropriate environment, it can form a well organized and oriented nerve trunk. In the mesothelial chambers, the regenerating nerve is surrounded by a loose cellular stroma and a small amount of interstitial fluid, which was found to contain trophic activity for cultured rodent sensory neurons. Such factors may also support nerve regeneration in vivo.

We present a procedure for the dissociation and growth in serum-free defined culture medium of 4-day chick embryo lumbar spinal cord (LC4) neurons. LC4 neurons will not survive for even 24 h without the addition of trophic supplements (putative spinal cord neuronotrophic factors, SCNTFs). Serum-free medium conditioned over chick embryo heart and skeletal muscle, mouse Schwann and rat RN22 Schwannoma cell cultures were found to contain SCNTF activity which could be quantitated using a convenient neuronal survival bioassay. RN22 conditioned medium also contains polyornithine-binding neurite promoting factors (PNPFs) which can be physically separated from SCNTF. When SCNTF and PNPF were presented to LC4 neurons individually or in combination (i) SCNTF, but not PNPF, supported neuronal survival whereas (ii) PNPF, but not SCNTF, induced neurite production. When LC4 neurons were grown in SCNTF alone, nearly all of them exhibited a flattened, circular, ‘fried-egg’ morphology. The subsequent addition of PNPF caused these cells to extend long neurites with characteristic terminal growth-cone-like structures.

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.