Nerve growth factor and cholinergic CNS neurons studied in organotypic brain slices. Implication in Alzheimer's disease?

Nerve growth factor (NGF) is a potent growth factor for cholinergic neurons. The aim of the present study was to investigate if NGF affects cholinergic neurons of the basal nucleus of Meynert (nBM) in organotypic brain slices. In single nBM slices cholinergic neurons rapidly degenerated when incubated without NGF. The number of remaining neurons was rescued by NGF application at any time point. When nBM slices were co-cultured with a cortex slice the number of cholinergic neurons was significantly increased pointing to a trophic influence of the cortex. Incubation with acetylcholine precursors did not affect the survival of cholinergic neurons. There was no significant difference when postnatal day 3 or day 10 nBM slices were cultured. In conclusion, NGF is the most potent growth factor for cholinergic neurons and is a promising candidate for treating Alzheimers disease, however, the delivery of NGF to the brain must the solved.     

Vascular endothelial growth factor counteracts NMDA-induced cell death of adult cholinergic neurons in rat basal nucleus of Meynert

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in the brain and has recently been shown to possess neuroprotective activity. The aim of the present study was to observe if VEGF can counteract the excitotoxic N-methyl-D-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert in vivo in adult rats. Immunohistochemistry was used to detect vascular structures (RECA-1 or laminin staining) and cholinergic neurons (choline-acetyltransferase). To compare the in vivo VEGF effects also in vitro, VEGF was applied to axotomized cholinergic neurons in organotypic brain slices with or without NMDA. Our data provide evidence that VEGF counteracted cell death in vivo in adult rats but did not protect cholinergic neurons in developing brain slices. Nerve growth factor protected cholinergic neurons in vivo as well as in vitro. In conclusion, VEGF exhibits neuroprotective activity on adult cholinergic neurons of the basal nucleus of Meynert.     

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.     

Reversible schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor

Substantial dysfunction and loss of cholinergic neurons occur in Alzheimer's disease (AD). Nerve growth factor (NGF) is a potent neurotrophic factor for cholinergic basal forebrain neurons, and the use of NGF to stimulate residual dysfunctional cells in AD is being considered. To define the effects of NGF on other cell populations in the brain, NGF was continuously infused into the lateral ventricle of rats for 7 weeks. At the end of treatment, Schwann cell hyperplasia and abundant sensory and sympathetic neurite sprouting were observed in the subpial region of the medulla oblongata and the spinal cord. Following withdrawal of NGF, the Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites disappeared completely. These findings suggest that better temporal and spatial delivery systems for NGF must be explored to limit potential undesirable side effects while maintaining the survival and function of diseased basal forebrain cholinergic neurons.     

Brain-derived neurotrophic factor administration protects basal forebrain cholinergic but not nigral dopaminergic neurons from degenerative changes after axotomy in the adult rat brain.

Cell culture studies with dissociated primary cultures from embryonic rat brain revealed that brain-derived neurotrophic factor (BDNF) promotes the developmental differentiation of both basal forebrain cholinergic and mesencephalic dopaminergic neurons. These studies suggested that, in the adult brain, BDNF may be able to protect cholinergic and dopaminergic neurons from degenerative changes induced by axotomy, similar to the known protective action of NGF in cholinergic neurons. Testing this hypothesis, we found that intraventricular administration of recombinant human BDNF (rhBDNF) to adult rats with transections of the fimbria significantly reduces axotomy-induced degenerative changes of the cholinergic cells in the basal forebrain. No such effect was seen on the dopaminergic neurons of the ventral mesencephalon after transection of their axons ascending in the medial forebrain bundle. Injected in equal amounts, rhBDNF and recombinant human NGF had quantitatively different effects on the cholinergic neurons. BDNF sustained only part of the population of cholinergic neurons affected by the lesion, whereas the entire population was protected by NGF treatment.     

Nerve growth factor increases the size of intracortical cholinergic transplants

The effects of continuous intracortical mouse Nerve Growth Factor on fetal rat basal forebrain transplants in denervated adult rat neocortex were investigated. Enzyme-linked immunoassay (ELISA) was used to measure the time course of endogenous NGF protein production in neocortex, hippocampus, and basal forebrain in a cohort of animals receiving unilateral ibotenic acid (IBO) lesions of the nucleus basalis magnocellularis (nBM). A second cohort of IBO-nBM lesioned animals received transplants of fetal basal forebrain followed by two to four weeks of continuous NGF or cytochrome-C infusion into the ipsilateral frontoparietal neocortex. To study the effects of abnormally high NGF doses on transplanted and host tissue, the cumulative dose of intracortical NGF was on the order of micrograms, compared with maximum picogram levels of neocortical NGF produced following IBO-nBM lesions. A four-fold increase in transplant size, and greater cell and fiber densities were observed in NGF-treated compared with NGF-untreated transplants. No adverse histological effects of long-term, high-dose NGF treatment were observed on transplanted basal forebrain or host neocortical tissue. These data indicate that cholinergic-rich mammalian brain tissue and intrinsic host tissue can be stimulated by high doses exogenous NGF without obvious deleterious effects.     

Nerve growth factor does not influence the expression of β amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies

We investigated the effect of NGF on amyloid precursor protein (APP) mRNA levels in the rat septal/nucleus basalis system. Total APP mRNA and APP 695 mRNA were determined in basal forebrain primary cell cultures exposed acutely and chronically to NGF (150–300 ng/ml) and, in vivo, in the septal area and striatum of rat pups after multiple intracerebroventricular injections of NGF. The trophic factor was able to affect cholinergic neurons in both paradigms, as evidenced by the significant increase of choline acetyltransferase (ChAT) activity induced by NGF in cell cultures (+80%) and in the striatum (+240%) of rat pups. In spite of this effect, no significant change of APP mRNA expression was observed in neuronal cultures and brain tissues. These data indicate that the neurotrophic effect of NGF on forebrain cholinergic neurons is not always associated with an alteration of APP expression.     

The effects of anti‐nerve growth factor monoclonal antibodies on developing basal forebrain neurons are transient and reversible

In order to reassess the role of nerve growth factor (NGF) on rat basal forebrain cholinergic neurons (BFCNs) survival and/or phenotype maturation during the early postnatal life, we immunoneutralized NGF in vivo . Hybridoma cells producing the neutralizing anti‐NGF monoclonal antibody αD11 were implanted in the lateral ventricle of the rat at different postnatal ages (P2, P8 and P15) and the effects on the number and the soma size of cholinacetyltransferase (ChAT) positive neurons were analysed 1, 2 or 3 weeks after the injection. A marked decrease in the number and in the soma size of BFCNs was observed implanting hybridoma cells at P2 and performing the analysis 1 week later. These effects are reversed 3 weeks after the implant of hybridoma cells at P2. At this time point, the levels of αD11 antibodies in the brain parenchyma are still in a vast molar excess over endogenous NGF. No effects on BFCNs were observed implanting αD11 cells at P15 while LGN neurons showed marked shrinkage. Our results demonstrate that the reduction in the number of ChAT‐positive neurons during the first two postnatal weeks of anti‐NGF treatment is not due to cell death. We conclude that NGF is not a survival factor for BFCNs, and that the influence of NGF on BFCNs cell maturation during the first 2 postnatal weeks is transient and reversible. Our results on tyrosine kinase (Trk) coexpression, suggest that NGF may cooperate with other factors in the cholinergic phenotype differentiation and maintenance after the second postnatal week.     

Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons

Nerve growth factor (NGF) has recently been implicated as a trophic agent in the survival and maintenance of basal forebrain cholinergic neurons. To test the hypothesis that NGF may play a role in the age-related degeneration of basal forebrain neurons and decline of cerebral cholinergic function, we have used a monoclonal antibody to the NGF receptor, 192 IgG, to immunocytochemically visualize and compare rat basal forebrain neurons responsive to NGF in aged (30 months) and young adult (10 months) rats. In a subpopulation of aged rats, NGF receptor-immunoreactive cells in the basal forebrain appear vacoulated and shrunken, and the neuropil staining is markedly reduced. While no substantial decline in cell density is apparent in Nissl-stained sections, the number of NGF receptor-positive cell profiles within the vertical limb of diagonal band nuclei is reduced by an average of 32% in aged rats. Marked reduction in the expression of NGF receptors in aged rats may signify loss of capacity of the basal forebrain neurons to bind and transport NGF from their terminals in the hippocampus and cortex, subsequent decrease in NGF delivered to the cell bodies, and eventual cellular dysfunction and death of neurons in aging.     

Transplantation of a temperature-sensitive, nerve growth factor-secreting, neuroblastoma cell line into adult rats with fimbria-fornix lesions rescues cholinergic septal neurons

The HT4 cell line was derived from infection of a mouse neuroblastoma cell line with a retrovirus that encoded the temperature-sensitive (ts) mutant of SV40 large T antigen. At nonpermissive temperature, HT4 cells differentiated with neuronal morphology, expressed neuronal antigens, synthesized nerve growth factor (NGF) mRNA, and secreted biologically active NGF in vitro. We sought to establish whether transplanted HT4 cells expressed class I major histocompatibility complex (MHC) antigens, a partial requirement for recognition by cytotoxic T lymphocytes (CTL), and thus be susceptible to xenograft rejection. Differentiated HT4 cells expressed marginally detectable levels of class I MHC antigens, but demonstrated higher levels of class I MHC expression after treatment with interferon-gamma. However, HT4 cells were resistant to direct lysis by perforin, the pore-forming protein of CTLs, and thus may have potential use in xenograft experiments. To address whether HT4 cells secrete NGF in vivo, HT4 cells were transplanted into adults rats with unilateral fimbria-fornix transections. A ts cell line derived from P4 cerebellum, BT1, that does not differentiate with neuronal phenotype or synthesize NGF in vitro, was transplanted as a control. Six weeks posttransplant. HT4 cells had integrated into host CNS without forming tumors. In BT1 transplants, the number of medial septal acetylcholinesterase (AChE)-positive cells was reduced to 26-39% of the contralateral control side, depending on the rostrocaudal level. In HT4 transplants, the number of cholinergic septal neurons was 58-78% of the contralateral side. This percentage was significantly (P less than 0.005) greater than that seen with BT1 transplants, indicating that transplanted HT4 cells secrete NGF in vivo and rescue cholinergic septal neurons following fimbria-fornix transection.     

Trophic actions of recombinant human nerve growth factor on cultured rat embryonic CNS cells

NGF is a neurotrophic factor for basal forebrain cholinergic neurons and may serve to counteract the cholinergic deficits that are observed in Alzheimer's disease. Prior to the introduction of clinical trials, it is essential that recombinant human NGF (rhNGF) be produced and that its actions on target cells in the CNS be demonstrated. We prepared rhNGF and examined its actions on fetal rat brain neurons in culture including, in particular, the cholinergic neurons of the basal forebrain. rhNGF was more potent in increasing choline acetyltransferase (ChAT) activity in septal cultures than NGF purified from mouse salivary glands (mNGF). ED50s of the beta-NGF dimers were 4.9 pM for rhNGF and 12.4 pM for mNGF. The maximal ChAT activity response was achieved at approximately 35 pM with both NGFs and their efficacies were not significantly different. The two NGFs were not additive in effect. Identical to the results with mNGF, rhNGF strongly enhanced the intensity of ChAT immunostaining in septal cultures. Neither rhNGF nor mNGF affected the appearance of the cultures under phase-contrast illumination. Survival of cells at very low plating density on polyornithine/laminin-coated culture dishes was not affected by rhNGF or mNGF. Protein content and the uptake of GABA were also unaffected. At concentrations of up to 10 micrograms/ml, rhNGF did not significantly increase uptake of dopamine into cultures of ventral mesencephalon. We conclude that rhNGF produces potent and selective actions on cholinergic neurons of the basal forebrain as previously shown for mNGF.     

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal-basal forebrain neurons: an in vitro analysis

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.     

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.     

Effects of intraventricular transplantation of NGF-secreting cells on cholinergic basal forebrain neurons after partial immunolesion

The aim of the present study was to examine the effects of nerve growth factor on brain cholinergic function after a partial immunolesion to the rat cholinergic basal forebrain neurons (CBFNs) by 192 IgG-saporin. Two weeks after intraventricular injections of 1.3 μg of 192 IgG-saporin, about 50% of CBFNs were lost which was associated with 40–60% reductions of choline acetyltransferase (ChAT) and high-affinity choline uptake (HACU) activities throughout the basal forebrain cholinergic system. Two groups of lesioned animals received intraventricular transplantations of mouse 3T3 fibroblasts retrovirally transfected with either the rat NGF gene (3T3^NGF+) or the retrovirus alone (3T3^NGF−) and were sacrificed eight weeks later. In vivo production of NGF by 3T3^NGF+ cells was confirmed by NGF immunohistochemistry on the grafts and NGF immunoassay on cerebrospinal fluid (CSF) samples. Both ChAT and HACU activities returned to normal control levels in the basal forebrain and cortex after 3T3^NGF+ transplants, whereas no recovery was observed in 3T3^NGF− transplanted animals. There was a 25% increase in the size of remaining CBFNs and an increased staining intensity for NGF immunoreactivity in these cells after NGF treatments. Acetylcholinesterase (AChE) histochemistry revealed that the optical density of AChE-positive fibers in the cerebral cortex and hippocampus were reduced by about 60% in immunolesioned rats which were completely restored by 3T3^NGF+ grafts. In addition, decreases in growth-associated protein (GAP)-43 immunoreactivity after immunolesion and increases in synaptophysin immunoreactivity after 3T3^NGF+ grafts were observed in the hippocampus. Our results further confirm the notion that transfected NGF-secreting cells are useful in long-term in vivo NGF treatment and NGF can upregulate CBFN function. They also highly suggest that NGF induces terminal sprouting from remaining CBFNs. © 1996 Wiley-Liss, Inc.     

Long-term effects of selective immunolesions of cholinergic neurons of the nucleus basalis magnocellularis on the ascending cholinergic pathways in the rat: A model for Alzheimer's disease

Alzheimer's disease is associated with a significant decrease in the cholinergic input to the neocortex. In a rat model of this depletion, we analyzed the subsequent long-term changes in cholinergic fiber density in two well-defined areas of the frontal and parietal cortices: Fr1, the primary motor cortex, and HL, the hindlimb area of the somatosensory (parietal) cortex, two cortical cholinergic fields that receive inputs from the nucleus basalis magnocellularis (nBM). A specific cholinergic lesion was induced by the intraparenchymal injection of 192 IgG-saporin into the nBM. Choline acetyltransferase (ChAT) immunohistochemistry was applied to identify the loss of cholinergic neurons in the nBM, while acetylcholinesterase (AChE) enzyme histochemistry was used to analyze the decreases in the number of cholinoceptive neurons in the nBM and the cholinergic fiber density in the Fr1 and HL cortical areas in response to the nBM lesion. The immunotoxin differentially affected the number of ChAT- and AChE-positive neurons in the nBM. 192 IgG-saporin induced a massive, irreversible depletion of the ChAT-positive (cholinergic) neurons (to 11.7% of the control level), accompanied by a less dramatic, but similarly persistent loss of the AChE-positive (cholinoceptive) neurons (to 59.2% of the control value) in the nBM within 2 weeks after the lesion. The difference seen in the depletion of ChAT- and AChE-positive neurons is due to the specificity of the immunotoxin to cholinergic neurons. The cholinergic fiber densities in cortical areas Fr1 and HL remained similarly decreased (to 62% and 68% of the control values, respectively) up to 20 weeks. No significant rebound in AChE activity occurred either in the nBM or in the cortices during the period investigated. This study therefore demonstrated that, similarly to the very extensive reduction in the number of ChAT-positive neurons in the nBM, cortical areas Fr1 and HL underwent long-lasting reductions in the number of AChE-positive fibers in response to specific cholinergic lesioning of the nBM.     

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.     

Expression of superoxide dismutase messenger RNA in adult rat brain cholinergic neurons

Superoxide dismutase (SOD) protects cells exposed to an excess of the free radical nitric oxide, by preventing the formation of peroxynitrite. Certain central cholinergic neurons express constitutive nitric oxide synthase (nNOS), and presumably they are at risk from peroxynitrite intoxication. Immunocytochemistry for choline acetyltransferase (ChAT) was combined with in situ hybridization histochemistry (ISHH) to examine whether brain cholinergic populations differ with respect to their expression of the messenger RNA molecules (mRNAs) for the manganese-dependent (Mn-SOD) and copper/zinc-dependent superoxide dismutases (Cu/Zn-SOD). The cholinergic neurons located in the reticular formation of the upper brainstem (the laterodorsal tegmental nucleus [LDTN] and the pedunculopontine nucleus [PPN]) were found to express relatively high levels of Mn-SOD mRNA, whereas cholinergic neurons located in the basal forebrain (substantia innominata [SI], diagonal band [DB], medial septum [MS], and the nucleus basalis magnocellularis [nBM]), and the striatal cholinergic interneurons expressed low to intermediate levels of Mn-SOD mRNA. The rank order of median Mn-SOD mRNA density per cholinergic cell was LDTN > PPN > SI > striatum = nBM = DB > MS. This is similar to the rank order of nNOS mRNA densities in the cholinergic cells in these regions (R=0.9, p<0.02). The rank order of Cu/Zn-SOD mRNA levels in cholinergic populations (DB > LDTN = PPN = MS > SI = nBM = striatum) was not correlated with nNOS mRNA (R = 0.29, P>0.05). Thus, for cholinergic neurons, Mn-SOD may be important for protection from NO-related oxidative stress.     

Altered NGF protein levels in different brain areas after immunolesion

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75^NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75^NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75^NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75^NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues. © 1996 Wiley-Liss, Inc.     

Intravenous infusion of nerve growth factor‐secreting monocytes supports the survival of cholinergic neurons in the nucleus basalis of meynert in hypercholesterolemia brown‐norway rats

The recruitment of monocytes into the brain has been implicated in Alzheimer's disease and recent studies have indicated that monocytes can reduce amyloid plaque burden. Our previous investigations have shown that hypercholesterolemic rats develop cognitive, cholinergic, and blood–brain barrier dysfunction, but do not develop amyloid plaques. This study was designed to evaluate the effects of repeated intravenous (i.v.) infusion (via the dorsal penile vein) of primary monocytes on cognition, the cholinergic system, and cortical cytokine levels in hypercholesterolemia Brown‐Norway rats. In addition, we also transduced the monocytes with nerve growth factor (NGF) to evaluate whether these cells could be used to deliver a neuroprotective agent to the brain. Our results indicate that repeated i.v. infused monocytes migrate into the brains of hypercholesterolemic rats; however, this migration does not translate into marked effects on learning. Animals receiving NGF‐loaded monocytes demonstrate slightly improved learning and significantly elevated cholinergic neuron staining compared to treatment with monocytes alone. Furthermore, our data indicate that repeated infusion of monocytes does not lead to elevated cytokine secretion, indicating that no inflammatory response is induced. This study provides an experimental attempt to evaluate the effects of blood‐derived primary monocytes in hypercholesterolemia rats. © 2013 Wiley Periodicals, Inc.     

Mouse nerve growth factor prevents degeneration of axotomized basal forebrain cholinergic neurons in the monkey

NGF, a trophic polypeptide, is necessary for the normal development and survival of certain populations of neurons in the CNS and PNS. In the CNS, cholinergic neurons of the basal forebrain magnocellular complex (BFMC) are prominent targets of NGF. During rat development, NGF increases the activity of ChAT in these neurons. In adult rats with experimental injury of axons in the fimbria-fornix, NGF prevents degenerative changes in axotomized cholinergic BFMC neurons in the medial septal nucleus (MSN). Because the amino acid sequences of NGF and its receptor (NGF-R) are highly conserved across species, we hypothesized that mouse NGF would also prevent degeneration of cholinergic BFMC neurons in nonhuman primates. Therefore, the present study was designed to test whether fimbria-fornix lesions result in retrograde degenerative changes in basal forebrain cholinergic neurons in macaques, whether these changes are prevented by mouse NGF, and whether the protective effect of NGF is selective for cholinergic neurons of the basal forebrain. Following unilateral complete transection of the fornix, animals were allowed to survive for 2 weeks, during which time half of the subjects received intraventricular NGF in vehicle and the other half received vehicle alone. In animals receiving vehicle alone, there was a 55% reduction in the number of ChAT-immunoreactive cell bodies within the MSN ipsilateral to the lesion; loss of immunoreactive somata was more severe in caudal planes of the MSN. Remaining immunoreactive neurons appeared smaller than those in control, unoperated animals. In Nissl stains, there was no apparent loss of basophilic profiles in the MSN, but cells showed reduced size and intensity of basophilia. Treatment with NGF almost completely prevented reductions in the number and size of cholinergic neurons and had a significant general effect in preventing atrophy in basophilic magnocellular neurons of the MSN, though some basophilic neurons in the MSN did not appear to respond to NGF. Adjacent 7-microns-thick sections stained with ChAT and NGF-R immunocytochemistry revealed that these markers are strictly colocalized in individual neurons in the MSN in controls and in both groups of experimental animals. Thus, mouse NGF profoundly influences the process of axotomy-induced retrograde degeneration in cholinergic BFMC neurons in primates. The in vivo effectiveness of mouse NGF on primate BFMC neurons suggests that mouse or human recombinant NGF may be useful in ameliorating the ACh-dependent, age-associated memory impairments that occur in nonhuman primates.(ABSTRACT TRUNCATED AT 400 WORDS)