Neuron-specific enolase,neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

Summary Immunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.     

Altered neuronatin expression in the rat dorsal root ganglion after sciatic nerve transection

Background  

Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among them, neuronatin (Nnat) was the one which expression was significantly up-regulated. Nnat was identified as a gene selectively expressed in neonatal brains and markedly reduced in adult brains. The present study investigated whether the expression of Nnat correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

Expression of phosphorylated high molecular weight neurofilament protein (NF-H) and vimentin in human developing dorsal root ganglia and spinal cord

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.     

Immunohistochemical localization of S-100 protein subunits (alpha and beta) in dorsal root ganglia of the rat

The distribution of S-100 protein and their subunits (alpha and beta) in lumbar dorsal root ganglia of adult rat was investigated immunohistochemically using monoclonal antibodies against the S-100 protein, alpha-subunit and beta-subunit of S-100 protein. The conventional S-100 protein antibody stained both neurons (large and intermediate in size; 20.3% and 41 +/- 3.2 microns of diameter) and glial cells (satellite cells and Schwann cells). The immunoreaction for the alpha-subunit was observed in the perikarya of some large and intermediate sized neurons (17.2%, 45.6 +/- 6.1 microns of diameter), satellite cells and Schwann cells, whereas the beta-subunit immunoreactivity was found principally in glial cells, and in a scarce number of large and intermediate sized neurons (2.8%, 43.3 +/- 5 microns of diameter) Our results demonstrate that a subpopulation of large and intermediate sized neurons of lumbar DRG contain alpha- and beta-subunits of S-100 protein, being alpha-subunit predominant. Furthermore, the satellite glial and Schwann cells contain also the two subunits but mainly beta-subunit. These data confirm previous studies about the presence of S-100 protein in neurons of the central and peripheral nervous system.     

Evidence for neuropeptide FF (FLFQRFamide) in rat dorsal root ganglia.

By using specific antibodies and radioimmunological and immunohistochemical methods, we here show that neuropeptide FF (NPFF) occurs in cervical and lumbar dorsal root ganglia cells. Levels in the ganglia were low because they were detectable only after colchicine treatment or after unilateral dorsal rhizotomy. Similar high-performance liquid chromatography profiles were obtained from dorsal root ganglia and spinal cord extracts, indicating that the NPFF-immunoreactivity in the dorsal root ganglia represented similar molecular forms to that in the spinal cord. Immunocytochemistry localized NPFF-immunoreactivity in small- and medium-sized cells. These data suggest that low levels of NPFF present in fine diameter primary afferent fibers could be involved in the treatment of nociceptive information from fore- or hindlimb.     

Prenatal development of the rat dorsal root ganglia

Summary This study describes three-dimensional aspects of the development and pseudo-unipolarization of neuroblasts and the maturation of satellite cells in prenatal rat dorsal root ganglia, using scanning electron microscopy, after removal of extracellular connective tissue components by trypsin digestion and HC1 hydrolysis.At 14 days of gestation, the vast majority of neurons are spindle-shaped or bipolar and only 3% are unipolar, while at 16 and 18 days this percentage has increased to 30% and 91%, respectively. The initial portions of the central and peripheral neuronal processes gradually approach each other and form a common initial portion. Finally, the cytoplasm of this common initial portion becomes thinner and elongates to form the stem process of the mature cell.Satellite cells are present from the beginning of the period studied, but intricate networks of branching satellite cell processes only develop after about day 17.     

State-dependent phosphorylation of epsilon-isozyme of protein kinase C in adult rat dorsal root ganglia after inflammation and nerve injury

The epsilon-isozyme of protein kinase C (PKCepsilon) and the vanilloid receptor 1 (VR1) are both expressed in dorsal root ganglion (DRG) neurons and are reported to be predominantly and specifically involved in nociceptive function. Using phosphospecific antibody against the C-terminal hydrophobic site Ser729 of PKCepsilon as a marker of enzyme activation, the state-dependent activation of PKCepsilon, as well as the expression of VR1 in rat DRG neurons, was evaluated in different experimental pain models in vivo. Quantitative analysis showed that phosphorylation of PKCepsilon in DRG neurons was significantly up-regulated after carrageen- and Complete Freund's Adjuvant-induced inflammation, while it was markedly down-regulated after chronic constriction injury. A double-labeling study showed that phosphorylation of PKCepsilon was expressed predominantly in VR1 immunoreactivity positive small diameter DRG neurons mediating the nociceptive information from peripheral tissue to spinal cord. The VR1 protein expression showed no significant changes after either inflammation or chronic constriction injury. These data indicate that functional activation of PKCepsilon has a close relationship with the production of inflammatory hyperalgesia and the sensitization of the nociceptors. Inflammatory mediator-induced activation of PKCepsilon and subsequent sensitization of VR1 to noxious stimuli by PKCepsilon may be involved in nociceptor sensitization.     

Ectopic discharges trigger sympathetic sprouting in rat dorsal root ganglia following peripheral nerve injury

Experimental backgrounds of ectopic discharges were made by i.p. administrating of 4-aninopyriding (4-AP), a K+ channel blocker, or anisodamine, a muscarinic receptor blocker, in CCI rats, and the sympathetic sprouting in the dorsal root ganglia (DRG) as well as the heat-hyperalgesia was observed. It was demonstrated that the increased ectopic discharges induced by 4-AP promote sympathetic sprouting in the DRG and a greater number of sympathetic basket cells were developed, causing exacerbation of heat-hyperalgesia in CCI rats. On the contrary, the sympathetic sprouting in the DRG and heat-hyperalgesia are evidently diminished after anisodamine injection. Our results suggest that ectopic discharges may be an immediate factor in triggering sympathetic sprouting in DRG following peripheral nerve injury.     

Female steroid hormones modulate receptors for nerve growth factor in rat dorsal root ganglia

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide, and it is primarily synthesized in dorsal root ganglia (DRG). Plasma CGRP levels increase during pregnancy and with steroid hormones, and nerve growth factor (NGF) stimulates calcitonin/CGRP promoter and CGRP synthesis in DRG. We previously showed that CGRP levels in DRG were stimulated with steroid hormone treatments in vivo but not in vitro. Thus, the stimulation of CGRP by these hormones may be indirect through the upregulation of NGF effects. We hypothesized that the female sex steroid hormones upregulate NGF receptors, trkA and p75(NTR), in DRG. We examined the effects of 17 beta-estradiol (E(2)) and progesterone (P(4)) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 microg E(2), 4 mg P(4), or 5 microg E(2) + 4 mg P(4) in 0.2 ml sesame oil or injected with oil only and were killed at 6, 24, and 48 h. In addition, ovx rats were also injected s.c. with varying doses (0.2, 1.0, 5.0, 25 microg) of E(2) (0.5, 1.5, 4, 10 mg) P(4), and (5 microg) E(2) + (0.5, 1.5, 4.0, 10 mg) P(4) in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E(2)) or 24 (for P(4) and E(2) + P(4)) h. Furthermore, groups of ovx rats were also killed at 12 and 24 h; 3 and 7 days; 2, 4, and 6 wk after ovariectomy. The DRGs were collected from all groups and then processed for Western immunoblotting to examine both trkA and p75(NTR) levels. Estradiol increased trkA at 6 h but not p75(NTR). Progesterone caused upregulation of trkA and p75(NTR) at 6 and 24 h. 17 beta-Estradiol + P(4) increased trkA at 6 and 24 h and p75(NTR) at all time points examined. One microgram of E(2) increased trkA but did not affect p75(NTR) levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P(4) increased p75(NTR). Five micrograms of E(2) coinjected with P(4) at 1.5 and 4 mg increased trkA, while p75(NTR) receptor was upregulated when coinjected with P(4) at 1.5 to 10 mg. The ovariectomy caused a decrease in trkA receptors compared to proestrus rats, and these decreases were significant by 6 wk, but surprisingly p75(NTR) increased at 2 wk after ovariectomy. 17 beta-Estradiol increased trkA but not p75(NTR) receptors in DRG, whereas P(4) caused increases in both trkA and p75(NTR) in DRG. In addition, the combination of these steroid hormones had more effect on both receptors than either hormone alone. Thus, we concluded that high levels of female steroid hormones such as those due to pregnancy or hormonal replacement therapy could increase NGF receptor expression in DRG that carry more NGF to elevate the CGRP synthesis in these groups. We suggested that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.     

Ectopic discharges trigger sympathetic sprouting in rat dorsal root ganglia following peripheral nerve injury

Experimental backgrounds of ectopic discharges were made by i.p. administrating of 4-aninopyriding (4-AP), a K+ channel blocker, or anisodamine, a muscarinic receptor blocker, in CCI rats, and the sympathetic sprouting in the dorsal root ganglia (DRG) as well as the heat-hyperalgesia was observed. It was demonstrated that the increased ectopic discharges induced by 4-AP promote sympathetic sprouting in the DRG and a greater number of sympathetic basket cells were developed, causing exacerbation of heat-hyperalgesia in CCI rats. On the contrary, the sympathetic sprouting in the DRG and heat-hyperalgesia are evidently diminished after anisodamine injection. Our results suggest that ectopic discharges may be an immediate factor in triggering sympathetic sprouting in DRG following peripheral nerve injury.     

Changes in the concentration of enolase isozymes and S-100 protein in degenerating and regenerating rat sciatic nerve

Levels of enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) and S-100 protein in rat sciatic nerves were determined during their degeneration and regeneration processes. The sciatic nerves were unilaterally crushed or severed. The rats were killed 1, 2, 6, and 8-9 weeks later, and both the proximal and distal portions of the damaged nerves were dissected. Control samples were obtained from the untreated contralateral hindlimbs. Enolase isozymes and S-100 protein in the nerve segments were determined with the enzyme immunoassay method. The control nerves contained about 40, 90, and 30 pmol/mg protein of alpha alpha, alpha gamma, and gamma gamma enolases, respectively, and 0.85 microgram/mg protein of S-100 protein. These levels were not affected by repetitive electrical stimulation of the nerve fibers in vivo. The levels of the nervous system-specific forms of enolase (alpha gamma and gamma gamma) and S-100 protein decreased markedly within a week in the distal portion of the crushed nerve (alpha gamma, 27 pmol/mg; gamma gamma, 5.5 pmol/mg; S-100 protein, 0.36 microgram/mg) with apparently no change in the concentration of alpha alpha enolase. These levels in the proximal portion of the crushed nerve remained unaltered. The sensory and motor functions impaired by the sciatic nerve crush showed a recovery more or less after 4-9 weeks. This recovery was accompanied by a gradual regaining of the specific proteins in the distal portion of injured nerves (alpha gamma, 64 pmol/mg; gamma gamma, 13 pmol/mg; S-100 protein, 0.63 microgram/mg at the 8-9th week).     

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express α2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.     

P0 and myelin basic protein-like immunoreactivities following ligation of the sciatic nerve in the rat

In this work we analyzed variations in the expression of MBPs and P₀ in ligated sciatic nerves of young and adult rats at 3, 7, and 14 days postligation (PL), by immunohistochemistry and SDS-PAGE of isolated myelin. A protein redistribution was seen in the distal stump of ligated nerves with the appearance of immunoreactive clusters. Using the KS400 image analyzer, immunostained area values were obtained from the different nerves dissected. In adult rats, there was an increase of the immunostained area for MBP from 3 to 7 days PL, coincident with a reorganization of the marker in clusters, followed by a marked decrease at 14 days. P₀ immunolabeling gave similar results without, however, a decrease of the immunostained area at the longer survival time tested. Young animals showed an acceleration in the process of protein redistribution and digestion within ligated nerves, which followed a similar pattern as that of adult animals. Analysis by electrophoresis showed a marked decrease in P₀ and MBP at 7 days PL in young rats and 14 days PL in adult rats. The functional significance of protein clustering within myelin in injured nerves deserves further analysis.     

Acetyl-cholinesterase and fluoride-resistant acid phosphatase activities in dorsal root ganglia in the rat

Acetylcholinesterase and fluoride-resistant acid phosphatase activities were contrasted in alternative serial sections of rat dorsal root ganglia. The morphometric analysis demonstrated no correlation between cellular size and enzymatic activity. Differences with previous works in this area are discussed.     

Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig

Summary Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.