Effect of yohimbine on nerve growth factor mRNA and protein levels in rat hippocampus

Activation of noradrenergic receptors has been shown to increase expression of nerve growth factor (NGF) gene in brain cells in vitro. The present studies were undertaken to determine if this stimulation was effective in vivo as well. Rats were administered the norepinephrine-releasing drug, yohimbine (YOH), and had their hippocampi assayed for NGF mRNA and protein at various times after the injection. It was found that yohimbine caused a 3-fold increase of NGF mRNA levels at 24 h. Protein levels, however, were unaltered at this time. Thus norepinephrine release in vivo appears to be sufficient for increasing mRNA level but not for translation to protein.     

Effect of substance P on proximal tubular reabsorption in the rat

Micropuncture and clearance techniques were used simultaneously to determine the effect of substance P on proximal tubular and overall renal function in anesthetized rats. This polypeptide, infused in saline at 50 pg/min into the abdominal aorta above the renal arteries, produced increases in urine flow, 2.7-3.7 mul/min.g kidney wt (P is less than 0.005); urinary sodium concentration, 32-61 meq/liter (P is less than 0.01); and sodium excretion, 89-223 neq/min (P is less than 0.005). Tubular fluid to plasma inulin concentration ratio measured in the last accessible proximal convolution fell from 2.21 to 1.80 (P is less than 0.001), and thus fractional reabsorption was reduced from 54 to 44% (P is less than 0.001). Absolute reabsorption by the proximal convoluted tubule was also reduced 15.5-12.5 nl/min (P is less than 0.025). In a control series of animals, saline alone infused at the same rate did not produce any statistically significant changes in the measured parameters over the same time period. The intrerenal mechanism responsible for the reduction in proximal reabsorption appears to be a tubular one since no consistent or significant changes were observed in kidney or single nephron glomerular filtration rate, renal plasma flow, or intrarenal hydrostatic pressures. No evidence was found to indicate redistribution of filtration rate, or plasma flow, or a reduction in filtration fraction.     

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.     

Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain

Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.     

P2X receptor-mediated excitation of nociceptive afferents in the normal and arthritic rat knee joint

OBJECTIVE: Platelet-derived growth factor (PDGF) induces cellular proliferation and differentiation by activating intracellular signaling mechanisms via their cognate receptors. In previous studies, we demonstrated that human brain tumors such as meningiomas, astrocytomas, medulloblastomas, and ependymomas expressed the messenger ribonucleic acid for the PDGF subunits and their receptors. In the present study, we investigated the expression of the messenger ribonucleic acid PDGF A and B chains and the PDGF alpha and beta receptors in 17 cases of oligodendrogliomas. METHODS: Measurements of messenger ribonucleic acid levels were obtained using radioactive complementary deoxyribonucleic acid probes. Protein expression was analyzed with specific antibodies. RESULTS: Sixteen of 17 tumors expressed the PDGF A subunit and all the PDGF alpha receptors. Furthermore, all the tumors expressed PDGF B and PDGF beta receptor subunits. CONCLUSION: The results of this study suggest that oligodendrogliomas may have an autocrine loop stimulated by the interaction of PDGF and its receptor simultaneously produced by these tumors.     

Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint

In view of recent evidence that nerves may be involved in bone formation, the present study examines the local occurrence of axons at the onset of secondary ossification center formation in the knee region of developing rats. Radiographic and histological examination showed that secondary ossification center formation commenced at day 10. At day 15 the epiphyseal ossification had reached a relatively mature state. As seen by light microscopy, cartilage canals first appeared at day 5, reaching the epiphyseal center by day 9. Axons exhibiting a neurofilament-like immunoreactivity emerged from the perichondrial plexa into the cartilage canals. Many calcitonin gene-related peptide (CGRP)-immunoreactive axons were found in the canals, as well as in the perichondrium. Axons with tyrosine hydroxylase-like immunoreactivity were not found in the canals, but such fibers occurred in relation to blood vessels at other sites. The canal-related axons disappeared between days 13 and 15, and the canals themselves did not persist beyond bone formation. As seen in the electron microscope, an individual canal contained 3-10 unmyelinated Schwann cell-enclosed axons with diameters of 0.1-2.0 microM. These observations show that putative sensory unmyelinated axons with CGRP-and SP-like immunoreactivity are transiently present during initiation of bone formation in developing epiphyses. Whether there is a causal relation between transient innervation and osteogenesis remains to be determined.     

Immunocytochemical co-localization of substance P and calcitonin gene-related peptide in afferent renal nerve soma of the rat

Substance P, calcitonin gene-related peptide and somatostatin immunoreactivities have been demonstrated in putative afferent renal nerve fibers in the rat. Utilizing retrograde-tracing and immunohistochemistry, we labeled afferent renal nerve soma throughout dorsal root ganglia T9 to L1. Most (85%) of afferent renal nerve perikarya were immunoreactive for calcitonin gene-related peptide, 21% had substance P immunoreactivity and none had somatostatin immunoreactivity. All renal afferents immunoreactive for substance P also contained calcitonin gene-related peptide. These results provide evidence that calcitonin gene-related peptide and substance P are present and co-localized in afferent renal nerves, and therefore, mediate transmission of afferent renal input to the spinal cord in the rat.     

Differential regulation of substance P by all members of the nerve growth factor family of neurotrophins in avian dorsal root ganglia throughout development

This study examined the effects of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 on substance P levels in dorsal root ganglia of the quail shortly after ganglia formation (stage 26, embryonic day 4.5), during the middle of development (stage 33, embryonic day 7.5) and during late development (stage 44, embryonic day 14). It has already been shown that nerve growth factor increases levels of substance P during the middle and late stages of development, and that messenger RNA for the neurotrophin receptors, trkA, trkB and trkC is present at all of these stages. Dorsal root ganglia were isolated, rinsed with defined medium to dilute endogenous neurotrophins and exposed to one of the neurotrophins for either 4 or 20 h. Substance P levels were quantitated using enzyme immunoassay. None of the neurotrophins had any effect on substance P levels in dorsal root ganglia obtained at stage 26 after either a 4 or 20 h exposure time. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 all significantly increased levels of substance P after either a 4 h or 20 h incubation in ganglia obtained at stages 33 and 44. The effects of nerve growth factor and neurotrophin-3 were specific: increases in substance P were completely blocked by simultaneous exposure to antibodies against either nerve growth factor or neurotrophin-3. The absence of any effect of neurotrophins on substance P expression during early development was unexpected, since dorsal root ganglia exhibit substantial levels of substance P and receptors for the neurotrophins are present and are apparently functional. It was also surprising that brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 induced increases in substance P levels during the middle and late stages of development, since substance P was thought to be exclusively localized to small TrkA neurons in dorsal root ganglia. However, immunocytochemical examination of dorsal root ganglia at stages 33 and 44 revealed substance P-like immunoreactivity in larger neurons as well as in small neurons. The results of this study have shown that different cellular responses to neurotrophins, such as effects on survival and/or peptide expression, may be acquired with differing temporal patterns not strictly related to expression of their receptors. Further, the regulation of neuropeptide synthesis in dorsal root ganglia is not due to any one neurotrophic factor. and the factors that regulate expression during early development are still unknown.     

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain

Stimulation of glucocorticoid or beta-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2-3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.     

Endogenous nerve growth factor regulates the sensitivity of nociceptors in the adult rat

Nerve growth factor (NGF) has a well characterized role in the development of the nervous system and there is evidence that it interacts with nociceptive primary afferent fibres. Here we applied a synthetic tyrosine kinase A IgG (trkA-IgG) fusion molecule for 10-12 days to the innervation territory of the purely cutaneous saphenous nerve in order to bind, and thereby neutralize endogenous NGF in adult rats. Using neurophysiological analysis of 152 nociceptors we now show that sequestration of NGF results in specific changes of their receptive field properties. The percentage of nociceptors responding to heat dropped significantly from a normal 57% to 32%. This was accompanied by a rightward shift and a reduced slope of the stimulus response function relating the intracutaneous temperature to the neural response. The number of nociceptors responding to application of bradykinin was also significantly reduced from a normal of 28% to 8%. In contrast, the threshold for mechanical stimuli and the response to suprathreshold stimuli remained unaltered, as did the percentage of nociceptors responding to noxious cold. The reduced sensitivity of primary afferent nociceptors was accompanied by a reduction in the innervation density of the epidermis by 44% as assessed with quantitative immunocytochemical analysis of the panaxonal marker PGP 9.5. This demonstrates that endogenous NGF in the adult specifically modulates the terminal arborization of unmyelinated fibres and the sensitivity of primary afferent nociceptors to thermal and chemical stimuli in vivo.     

Elevated nerve growth factor levels in the synovial fluid of patients with inflammatory joint disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean +/- SEM NGF concentration in normal synovial fluids was 95 +/- 33.2 pg/ml (range 39.1-143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 +/- 123.8 pg/ml (range 152-1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 +/- 90 pg/ml; range 89-1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Effect of combined administration of insulin-like growth factor and platelet-derived growth factor on the regeneration of transected and anastomosed sciatic nerve in rats

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.     

Intraoperative monitoring of the function of the peroneal nerve in knee joint operations

Operations on the knee are known to be associated with postoperative neurological complications. There is no consensus opinion on the causes of these complications. The aim of the present study was to develop a method for the intraoperative monitoring of the function of the common peroneal nerve. This was done as to identify intraoperative factors that might be responsible for reversible and irreversible neurological deficits. Computer-aided neuromonitoring is based on online digitizing of the surface EMG of the anterior tibial muscle. An algorithm continuously modifies the amplitude to determine the motor threshold. The method described has been used in 18 patients undergoing high tibial osteotomy. In 10 of the 18 patients, the nerve is rendered completely non-excitable after an average tourniquet application time of 59 min. This non-excitability was reversed on release of the tourniquet. In the remaining 8 patients, excitability was maintained throughout the ischaemic period, which did not exceed 60 minutes in any of the cases. Our method enables accurate quantification of the neural function throughout the entire operation, and convincingly documents the influence of ischaemia on peripheral nerve block.     

Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury

Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury.     

Regional distribution of substance P in the brain of the rat

Using a sensitive radioimmunoassay we have studied the regional distribution of substance P. The level of substance P is higher in the mesencephalon, hypothalamus and preoptic area than in other regions of the brain. Substance P is found in especially high concentrations in the reticular part of the substantia nigra and the interpeduncular nucleus. It is present in large amounts in several septal, preoptic and hypothalamic nuclei as well.     

Endogenous glutamate levels regulate nerve growth factor mRNA expression in the rat dentate gyrus

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.     

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.