Nerve Growth Factor Messenger RNA Research Articles

La relation avec le (la) patient(e) douloureux (se) chronique : comment relever le défi ? Questions posées à Madame Cécile Cunin-Roy

The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.

Expression of nerve growth factor and its receptors in the uterus of rabbits: functional involvement in prostaglandin synthesis

The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.

Dienogest inhibits nerve growth factor expression induced by tumor necrosis factor-α or interleukin-1β

Dienogest (DNG), a selective P receptor (PR) agonist, is used to treat endometriosis. To investigate whether DNG affects nerve growth factor (NGF) expression, we stimulated human endometrial epithelial cells (hEECs) with inflammatory cytokines. Prospective basic research study using immortalized hEEC lines. Development Research, Mochida Pharmaceutical Co., Ltd., Japan. None. Not applicable. In immortalized hEECs, NGF production induced by tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) was evaluated in the presence or absence of the synthetic progestin DNG or endogenous P. The NGF messenger RNA (mRNA) and protein were measured using real-time reverse transcriptase-polymerase chain reaction (PCR) and ELISA, respectively. The NGF bioactivity in the culture medium was measured by assaying neurite outgrowth of PC-12 cells. Tumor necrosis factor-α and IL-1β induced NGF mRNA and protein and increased NGF bioactivity in the culture medium. These activities were inhibited by DNG in a hEEC line that stably expresses PR. In contrast, in an hEEC line that constitutively expresses faint levels of PR, no inhibitory effect of DNG on NGF mRNA was detected. The NGF mRNA was also inhibited in hEEC lines that express only PR-A or only PR-B. Nerve growth factor is one of the key mediators that generates the pain associated with endometriosis. Dienogest inhibits NGF expression through PR-A and PR-B in hEEC, which may contribute to the pharmacological mechanisms of how DNG relieves pain in endometriosis.

Expression Profile of Nerve Growth Factor after Muscle Incision in the Rat

Previous studies have demonstrated that nerve growth factor (NGF) is an important mediator of pathologic pain. Many studies have focused on cutaneous mechanisms for NGF-induced hyperalgesia; few have examined its contribution in deeper tissues like muscle. This study examined pain behaviors and the expression of NGF in incised hind paw flexor digitorum brevis muscle. Adult Sprague-Dawley rats were pretreated with anti-NGF peptibody and underwent skin or skin plus deep fascia and muscle incision. Guarding pain behaviors were measured. Muscle NGF messenger RNA (mRNA) was measured by reverse-transcriptase polymerase chain reaction. Changes in NGF protein expression were measured using Western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. In situ hybridization for NGF mRNA was also performed. Pretreatment with anti-NGF peptibody (100 mg/kg) decreased the guarding behavior caused by deep fascia and muscle incision. Muscle NGF mRNA increased abruptly 2 h after incision and was the same as control by postoperative day 1. NGF protein increased from 4 h after incision and was sustained for several days. NGF was localized in many calcitonin gene-related peptide-positive axons, few N52-positive axons, but not isolectin B4-positive axons in incised muscle. The sources of NGF mRNA included keratinocytes in epidermis and fibroblasts in deeper tissues. Fibroblasts adjacent to the injury are sources of NGF in incised muscle. NGF is upregulated by incision of muscle and contributes to guarding pain behavior.

Nerve Growth Factor Expression after Plantar Incision in the Rat

Postoperative pain control remains a significant problem. Advances will proceed if we can further reveal the underlying mechanisms of incisional pain and its mediators. Previous studies have demonstrated that nerve growth factor (NGF) is released in incised tissue and contributes to hyperalgesia in incisional pain. The purpose of this study is to examine the expression of NGF in skin after planter incision. Adult Sprague-Dawley rats underwent incision at the plantar aspect of hind paw. The NGF messenger RNA (mRNA) was measured at various times after incision by polymerase chain reaction. NGF protein expression was detected by Western blot and immunohistochemistry in incisions. NGF mRNA increased from 2 to 4 h after incision and was the same as control by postoperative day 1. A large-molecular-weight form of NGF, approximately 75 kd, was found in normal skin. The large-molecular-weight NGF protein increased 4 h after incision and returned to baseline on postoperative day 7. The skin immediately adjacent to the incision had the greatest NGF expression. Immunohistochemical staining for NGF was present adjacent to the incision and localized in Schwann cells and axons. NGF mRNA is increased and a large-molecular-weight form of NGF protein is expressed in the region adjacent to the incision. NGF immunoreactivity is present in nerve bundles; both Schwann cells and axons are labeled. Immunoreactive NGF in axons is likely taken up into cut axons. This study suggests some common mechanisms for neuropathic and incisional pain.

Nerve Growth Factor Increases in Pancreatic β Cells After Streptozotocin-Induced Damage in Rats

We investigated short-term in vivo and in vitro effects of streptozotocin (STZ) on pancreatic beta cells. Male Wistar rats were treated with 75 mg/kg STZ, and, after 4 hrs blood glucose and insulin were measured and islet cells were isolated, cultured for 16 hrs, and challenged with 5.6 and 15.6 mM glucose. Treated rats showed hyperglycemia (approximately 14 mM) and a 70% decrease in serum insulin levels as compared with controls. Although insulin secretion by isolated beta cells from STZ-treated rats was reduced by more than 80%, in both glucose concentrations, nerve growth factor (NGF) secretion by the same cells increased 10-fold. Moreover, NGF messenger RNA (mRNA) expression increased by 30% as compared with controls. Similar results were obtained in an in vitro model of islet cells, in which cells were exposed directly to STZ for 1, 2, and 4 hrs and then challenged for 3 hrs with the same glucose concentrations. Our data strongly suggest that an early increase in NGF production and secretion by beta cells could be an endogenous protective response to maintain cell survival and that diabetes mellitus may occur when this mechanism is surpassed.

Expression of nerve growth factor messenger RNA in pancreatic cancer tissues in Sprague Dawley rats

Expression of nerve growth factor messenger RNA in pancreatic cancer tissues in Sprague Dawley rats

Nerve growth factor mediates alterations of colonic sensitivity and mucosal barrier induced by neonatal stress in rats

Background & Aims: Maternal deprivation (MD) increases nerve growth factor (NGF) expression and colonic mast cell density and alters visceral sensitivity. This study aimed to establish whether NGF overexpression induced by neonatal stress is involved in altered visceral sensitivity and gut mucosal integrity in adult rats. Methods: Male Wistar rat pups were either submitted to MD and treated with anti-NGF antibodies or left with their dam and treated daily with NGF. All rats were tested 10 weeks later for visceral sensitivity and 12 weeks later for gut permeability, myeloperoxidase activity, and mast cell numbers. Colonic NGF and NGF receptor expression were determined at 14 days and 12 weeks of age. To determine the involvement of colonic NGF overexpression and mast cell hyperplasia in visceral hyperalgesia induced by MD, neonatally deprived adult rats received anti-NGF antibodies or doxantrazole. Results: MD increased visceral sensitivity to rectal distention, gut permeability, colonic myeloperoxidase activity, and mast cell density, and anti-NGF antibodies abolished these effects. Neonatal daily treatment with NGF mimicked the alterations induced by MD on both rectal sensitivity and mucosal barrier. In deprived compared with nondeprived rats, colonic NGF immunostaining and NGF messenger RNA were increased at 14 days and 12 weeks. Overexpression of NGF receptor messenger RNA, present at 14 days, was not observed later. Moreover, adult deprived rats treated with doxantrazole or anti-NGF antibodies exhibited normal gut permeability and visceral sensitivity to rectal distention. Conclusions: These data indicate that NGF triggers and maintains long-term alterations of visceral sensitivity and gut mucosal integrity induced by MD.

Increased serum concentration of nerve growth factor in patients with microprolactinoma

Nerve growth factor (NGF) is known to play a role as a circulating neurokine, integrating signals from the neuro-immuno-endocrine system. The ability of NGF to activate the pituitary–adrenocortical axis, together with the increase of its serum concentration in pregnancy and lactation, supports the hypothesis that NGF is secreted by the pituitary gland and plays a role as modulator of endocrine functions. Evidence obtained both in vitro and in vivo in experimental animal models suggests that lactotroph cells secrete both prolactin (PRL) and NGF. Furthermore, in previous studies we demonstrate that cell lines derived from dopamine (DA)-sensitive human prolactinomas express high levels of NGF messenger RNA and protein. On these basis, we studied serum NGF concentrations in female patients with microprolactinoma ( n=4) and in control women ( n=5). PRL and NGF were measured at the diagnosis, during the thyrotropin releasing hormone (TRH) test and after the therapy with DA D2 receptor agonist cabergoline (0.25 mg, twice a week). Results obtained suggested that hyperprolactinemia (70.3 ± 8.4 ng/ml) paralleled markedly higher NGF levels (58.4 ± 18.7 pg/ml) compared to controls (PRL 8.7 ± 3.2 ng/ml, NGF 8.4 ± 1.3 pg/ml). Serum concentrations of NGF and PRL during the TRH test were closely associated ( r=0.943, p<0.01). Cabergoline therapy normalized PRL (7.9 ± 3.6 ng/ml) and induced a significant decrease of NGF levels (12.5 ± 4.9 pg/ml). In conclusions, data reported here indicated that, in human microprolactinomas, NGF is released in the bloodstream paralleling PRL-secretion and it is modulated by a neurotransmitter-regulated mechanism, since the normalization of PRL elicited by the DA D2 receptor agonist cabergoline induced a significant decrease of serum NGF as well.

Human dermal microvascular endothelial cells produce nerve growth factor: implications for wound repair.

Following cutaneous injury, sensory nerves regenerate into the dermis and epidermis. Tissues that are innervated by sensory nerves synthesize neurotrophins such as nerve growth factor (NGF). The close anatomic proximity of nerves and capillaries throughout the skin suggests that mutual regulation may exist between nerve fibers and microvascular endothelial cells (MECs) during wound repair. Release of the neuropeptide substance P by sensory nerves induces endothelial cell rounding, capillary leak, and cytokine upregulation. We propose that dermal endothelial cells produce neurotrophins required for nerve fiber maintenance and regeneration. In this study, we demonstrate that substance P stimulates NGF messenger RNA expression by cultured human dermal MECs. Likewise, enzyme-linked immunosorbant assay demonstrated that conditioned medium from cultured dermal MECs contains NGF. NGF bioactivity in the supemates was verified by conditioned medium-induced clonal rat pheochromocytoma (PC-12) cell differentiation. This activity was inhibited by anti-NGF antibodies. Therefore, we have demonstrated that substance P, an inflammatory neuropeptide released by sensory nerve fibers, induces endothelial cells to produce NGF. Our data suggest that MECs may be unrecognized contributors to nerve regeneration after cutaneous injury.

Human eosinophils produce neurotrophins and secrete nerve growth factor on immunologic stimuli

AbstractNeurotrophins, such as nerve growth factor (NGF) and neurotrophin-3 (NT-3), are essential for development, function, and survival of peripheral sympathetic and sensory neurons. Most eosinophilic leukocytes in the human body are localized in mucosal tissues; however, the roles of eosinophils in human diseases are not fully understood. We found that human eosinophils constitutively express messenger RNA for NGF and NT-3, synthesize and store these proteins intracellularly, and continuously replenish them. Incubation of eosinophils with a transcription inhibitor, actinomycin D, for 8 hours completely depletes intracellular NGF and NT-3. New synthesis of NGF is enhanced by Fc-receptor–mediated stimuli, such as immunoglobulin (Ig)A and IgG immune complexes; in contrast, production of NT-3 is not affected by these stimuli. Notably, supernatants of eosinophils stimulated with IgA immune complex and interleukin 5 promote neurite extension of the PC-12 pheochromocytoma cell line; this effect is abolished by pretreatment of the supernatants with anti-NGF–neutralizing antibody. By enzyme-linked immunosorbent assay, substantial amounts of NGF protein are also detected in the supernatants of stimulated eosinophils. Furthermore, in patients with seasonal allergic rhinitis, the concentrations of NGF in nasal secretions correlate with the magnitudes of eosinophilic inflammation in the airway, suggesting a potential clinical implication of eosinophil NGF. Our observations propose a new pathologic mechanism by which eosinophils may contribute to enhanced neurologic responses in patients with allergic diseases and other eosinophilic disorders. Alternatively, eosinophils may play important roles in maintenance and restoration of homeostatic functions of mucosal tissues through the pleitropic activities of NGF.

Estrogen and neuroinflammation

Women have a higher incidence of inflammatory disorders than men and also appear to perceive painful stimuli differently. It has been suggested that neuroinflammation plays a role in painful bladder disorders of uncertain etiology, such as interstitial cystitis. Nerve growth factor (NGF) is a neurotrophin produced in peripheral tissues that can also mediate pain and inflammation. We found that treatment of mice with the estrogen antagonist ICI 182,780 had no effect on bladder NGF content but decreased bladder NGF messenger RNA. Using immunohistochemistry, we demonstrated that the mucosa is the primary source of NGF in the mouse bladder, and the bladder mucosa also expresses estrogen receptor (ER)-α, ER-β, and the high-affinity NGF receptor tyrosine kinase A. Estrogen may also modulate neurogenic inflammation by interaction with other substances and cells that participate in the pathogenesis of neurogenic inflammation, including substance P, bradykinin, and mast cells. Collectively, these observations indicate that estrogen has the capacity to influence the onset and course of neurogenic inflammation of the bladder.

Differential patterns of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNA and protein levels in developing regions of rat brain.

The present studies were undertaken to characterize the regional and temporal patterns of neurotrophin messenger RNA and protein levels for beta-nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the developing CNS. We have examined the levels of these neurotrophin messenger RNAs with ribonuclease protection assays and corresponding protein levels with enzyme-linked immunosorbent assays in the developing Long-Evans rat hippocampus, neocortex and cerebellum on postnatal days 1, 7, 14, 21, and 92. In addition, immunohistochemistry was used to localize the neurotrophins in these developing brain regions. Results indicated that in neocortex and hippocampus, messenger RNA for both nerve growth factor and brain-derived neurotrophic factor increased in an age-dependent manner, reaching a plateau by postnatal day 14. In the neocortex, nerve growth factor and brain-derived neurotrophic factor protein levels both peaked at postnatal day 14. In hippocampus, nerve growth factor protein peaked at postnatal day 7 while brain-derived neurotrophic factor peaked at postnatal day 14. In cerebellum, nerve growth factor messenger RNA levels were flat, while nerve growth factor protein peaked at postnatal day 7. Brain-derived neurotrophic factor messenger RNA increased in an age-dependent manner while the pattern for its protein levels was mixed. Neurotrophin-3 messeger RNA levels increased in an age-dependent manner in hippocampus, peaked at postnatal day14 in cerebellum, and no changes occurred in neocortex. Neurotrophin-3 protein was at its peak at postnatal day 1 and thereafter decreased at other postnatal days in all three brain regions. Results of neurotrophin immunohistochemistry often paralleled and complemented enzyme-linked immunosorbent assay data, demonstrating specific cell groups containing neurotrophin proteins in these regions. Within each region, patterns with regard to messenger RNA and respective protein levels for each neurotrophin were unique. No consistent relationship between patterns of neurotrophin messenger RNAs and their cognate proteins was observed between regions. The different regional patterns for neurotrophin messengerRNA and protein levels in each brain region indicate that messenger RNA studies of neurotrophin messenger RNA must be augmented by protein determination to fully characterize spatial and temporal neurotrophin distribution.

Nerve growth factor but not neurotrophin-3 is synthesized by hippocampal GABAergic neurons that project to the medial septum

Conventional uptake of neurotrophins takes place at axon terminals via specific receptors, and is followed by retrograde transport. Recent studies demonstrated that, with the exception of nerve growth factor, other neurotrophins may be delivered anterogradely to the region containing the receptor expressing neurons. In this study we used a triple labeling method that combines retrograde tract tracing, in situ hybridization and immunocytochemistry to examine whether non-principal cells projecting from the hippocampus to the septum synthesize nerve growth factor. Our results show that, on average, 59% of the horseradish peroxidase-labeled hippocamposeptal nonpyramidal neurons also display nerve growth factor messenger RNA hybridization signal. The ratio was slightly higher in the CA1 stratum oriens and the hilus of the dentate gyrus (64 and 62%, respectively) compared to stratum oriens of the CA3 region (58%). In addition, we demonstrated that many nerve growth factor-positive septally projecting neurons also contain the calcium-binding protein calbindin d-28K, whereas nerve growth factor-negative projecting cells mostly lack this neurochemical marker. In contrast to nerve growth factor, neurotrophin-3 has never been found in hippocamposeptal cells. Hippocamposeptal GABAergic cells are reciprocally connected with the medial septum, thus they are in a key position to regulate nerve growth factor release as a function of the activity level in the septohippocampal system. Furthermore, our results raise the intriguing possibility that nerve growth factor may be transported also in an anterograde manner. Regardless of the direction of transport, the presence of nerve growth factor in hippocamposeptal cells suggests that long distance fast synaptic mechanisms and slow neurotrophin action are coupled in these neurons.

Traumatic injury induces differential expression of cell death genes in organotypic brain slice cultures determined by complementary DNA array hybridization

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, α-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1β isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, β 2 microglobulin, bax, bcl xl, brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1β, interleukin-6, tumor necrosis factor-α, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.

Human neoplastic Schwann cells: changes in the expression of neurotrophins and their low‐affinity receptor p75

Neurotrophins are known to influence Schwann cells during development and to promote peripheral nerve regeneration after axonal damage. In neoplastic conditions. Schwann cells from experimentally-induced schwannomas appear to retain their responsiveness to nerve growth factor (NGF), although the role of neurotrophins in the neoplastic process in poorly understood. In this study, human neoplastic Schwann cells (five cases of acoustic schwannoma and two cases of malignant peripheral nerve sheath tumours [MPNST]) were investigated for the expression in situ of molecules of the neurotrophin system. In particular, we studied the 75 kDa low-affinity receptor (p75) and the mRNA for its ligands, NGF and neurotrophin-3 (NT-3). By immunohistochemistry, the p75 receptor was found to be the present at high levels in Schwann cells from acoustic schwannomas, whereas it was very weak or absent in MPNST. Messenger RNA for NGF and NT-3 was detected by reverse transcriptase in situ polymerase chain reaction technique and showed the same fluctuation of p75, being up-regulated in acoustic schwannomas and very weak or absent in MPNST. In normal non-neoplastic tissue, no detectable amounts of either ligand or receptor were observed. Our results indicate that changes in the expression of neurotrophins and their p75 receptor occurred during the neoplastic transformation of Schwann cells. In benign schwannomas, such changes are likely to reflect the loss of axonal contact, while in MPNST they may be related to a complete derangement of cell machinery in the tumour cells.

Selegiline enhances NGF synthesis and protects central nervous system neurons from excitotoxic and ischemic damage

It has been previously demonstrated that selegiline, an irreversible monoamine oxidase B (MAO-B) inhibitor, potentiates glial reaction to injury and possesses some ‘trophic-like’ activities which do not depend on the inhibition of MAO-B and which are probably associated with the induction of astrocyte-derived neurotrophic substances. Based on these findings, we tried to find out whether selegiline is able to modify the expression of nerve growth factor (NGF) and to protect central nervous system (CNS) neurons from excitotoxic and ischemic damage. Selegiline (10 pM–1 nM) induced NGF messenger RNA (mRNA) expression in cultured rat cortical astrocytes as determined by reverse transcription-polymerase chain reaction (RT-PCR) followed by a corresponding increase in NGF protein content measured by two-site NGF-enzyme-linked immunosorbent assay (ELISA) in astrocyte-conditioned medium. Additionally, exposure of hippocampal cultures containing neuronal and glial cells to this drug at the same concentrations enhanced significantly the content of NGF measured in the culture medium after 6 h of incubation. We hypothesize that selegiline could rescue hippocampal neurons from injury by induction of astrocyte-derived NGF in this cell culture system. To test this hypothesis, an excitotoxic damage was induced in the same type of cells by exposure to 0.5 mM l-glutamate for 1 h. Selegiline (10 pM–1 nM) present in the growth medium 6 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement) protected hippocampal neurons from excitotoxic death. Furthermore, administered intraperitoneally (i.p.) (8 × 15 mg/kg per day) this drug enhanced the expression of NGF message in intact rat cerebral cortex and protected rat cortical tissue from ischemic insult due to permanent occlusion of the middle cerebral artery (MCA). The neuroprotective activity of selegiline (5 × 10 mg/kg per day i.p.) was also demonstrated in a mouse model of focal cerebral ischemia. The present data show that selegiline induced NGF expression in cultured rat cortical astrocytes. In mixed primary cultures of hippocampal neuronal and glial cells, selegiline increased NGF protein content and protected hippocampal neurons from excitotoxic degeneration. In vivo, this drug induced NGF gene expression in cerebral cortex from intact rats and protected rat and mouse cortical tissue from ischemic insult after occlusion of the MCA. Our results indicate that the induction of astrocyte-derived NGF could contribute to the neuroprotective activity of selegiline demonstrated both in vivo and in vitro and can explain, in part, the ‘trophic-like’ properties of this compound which has been observed by others.

Cell-specific modulation of basal and seizure-induced neurotrophin expression by adrenalectomy.

Reports of glucocorticoid effects on neurotrophin expression suggest that adrenal hormones may contribute to the pattern of changes in the expression of these factors induced by neuronal activity and seizures. To examine this possibility, the present study evaluated the influence of adrenalectomy on basal expression and seizure-induced alterations in levels of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 messenger RNAs in hippocampus, entorhinal cortex, and superficial neocortex. For determination of hormone effects on basal expression, adult male rats were adrenalectomized and killed 10-14 days later with paired adrenal-intact controls. For studies of adrenal steroid involvement in expression following seizure, adrenalectomized and adrenal-intact rats received a seizure-producing lesion of the dentate gyrus hilus. Changes in neurotrophin messenger RNA content were assessed by quantitative in situ hybridization. Adrenalectomy alone had no significant effect on brain-derived neurotrophic factor messenger RNA content but did result in cell-specific decreases in nerve growth factor and neurotrophin-3 messenger RNAs. Nerve growth factor messenger RNA levels were reduced in hippocampal stratum granulosum, entorhinal cortex, and neocortex but not in cells of the hippocampal molecular layers or hilus. With adrenalectomy, neurotrophin-3 messenger RNA was virtually eliminated from CA2 stratum pyramidale, partially reduced in stratum granulosum, but unaffected in neurons of the hippocampal molecular layers or entorhinal cortex. These effects were partially reversed by corticosterone (2 mg/l) supplement to the drinking saline. In experimental-seizure rats, adrenalectomy did not alter the direction or basic pattern of seizure-induced changes in neurotrophin expression but did change the time courses and magnitudes of these effects. In all areas measured, brain-derived neurotrophic factor messenger RNA content was more greatly and persistently elevated by seizure in adrenalectomized as compared with adrenal-intact rats. In contrast, with adrenalectomy seizures induced smaller increases in nerve growth factor messenger RNA content. Adrenalectomy augmented the decrease in neurotrophin-3 messenger RNA induced by seizure in hippocampus but not in entorhinal cortex. These results demonstrate that adrenal hormones play a major role in the regulation of basal nerve growth factor and neurotrophin-3 messenger RNA expression by specific populations of forebrain neurons. Moreover, the adrenal steroids have opposite effects on activity-dependent changes in brain-derived neurotrophic factor and nerve growth factor messenger RNA levels but are not required for the basic pattern of changes in neurotrophin messenger RNA expression elicited by recurrent seizures.

Changes in nerve growth factor and preprotachykinin messenger RNA levels in the iris and trigeminal ganglion in diabetic rats; effects of treatment with insulin or nerve growth factor

This study was designed to explore effects of experimental diabetes mellitus on expression of substance P in the trigeminal ganglion and of nerve growth factor (NGF) in the iris. Rats with streptozotocin-diabetes showed an increase in NGF mRNA in the iris (P < 0.01) which was corrected by insulin treatment. There was also an increase in the levels of mRNA for the substance P precursor, preprotachykinin (PPT), in the trigeminal ganglion of these animals, despite there being no change in GAP-43 mRNA. Since expression of both of these peptides is sensitive to NGF in vitro, we examined the effect of treatment of diabetic rats with NGF at three different doses (0.2, 0.5 and 1.0 mg/kg body weight). There was a dose-dependent increase in both gamma-PPT and GAP-43 mRNA in the trigeminal ganglia of NGF-treated diabetic rats. The findings indicate that increased NGF may be responsible for raised substance P levels in the iris.

Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor messenger RNA in streptozotocin-diabetic rats: Effects of insulin treatment

The role of sub-optimal neurotrophic support in the aetiology of the sensory neuron dysfunction associated with diabetic neuropathy was investigated. The status of sciatic nerve neuropeptide content was related to target tissue nerve growth factor messenger RNA levels in streptozotocin-diabetic rats. The levels of substance P and calcitonin gene-related peptide in diabetic sciatic nerve were significantly lowered by approximately 50% and 28%, respectively, compared with aged matched controls and insulin-treated diabetic rats ( P < 0.01) for both peptides and both comparisons). Measurements of nerve growth factor messenger RNA levels in sensory neuron target tissues, namely foot-skin and soleus muscle, revealed deficits of approximately 50% in diabetic rats, with insulin treatment reversing the decrease in foot-skin but not in soleus muscle. The results show a possible correlation between deficient neuropeptide gene expression in sensory neurons and reduced nerve growth factor messenger RNA levels in target tissue.