Localization of CGRP and VEGF mRNAs in the mouse superior cervical ganglion during pre- and postnatal development.

The neuropeptide calcitonin gene-related peptide (CGRP) mediates inflammation and head pain by influencing the functional vascular blood supply. CGRP is a well-characterized mediator of receptor-regulated neurotransmitter release. However, knowledge regarding the role of CGRP during the development of the superior cervical ganglion (SCG) is limited. In the present study, we observed the localization of CGRP and vascular endothelial growth factor (VEGF-A) mRNAs during prenatal development at embryonic day 14.5 (E14.5), E17.5 and postnatal day 1 (P1) using in situ hybridization. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels were detected at E17.5. In contrast, the antisense probe for VEGF-A was detected by in situ hybridization in gradually increasing intensity from E14.5 to P1. The differences in the expression of these two markers revealed specific characteristics related to CGRP concentration and release compared to those of VEGF-A during development. The correlation between CGRP and VEGF-A may influence functional stress and the vascular blood supply during prenatal and postnatal development.

Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix.

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation.

BACKGROUND: Nerve growth factor (NGF) is the prototypical target-derived neurotrophic factor required for sympathetic neuron survival and for the growth and ramification of sympathetic axons within most but not all sympathetic targets. This implies the operation of additional target-derived factors for regulating terminal sympathetic axon growth and branching. RESULTS: Here report that growth differentiation factor 5 (GDF5), a widely expressed member of the transforming growth factor beta (TGFß) superfamily required for limb development, promoted axon growth from mouse superior cervical ganglion (SCG) neurons independently of NGF and enhanced axon growth in combination with NGF. GDF5 had no effect on neuronal survival and influenced axon growth during a narrow window of postnatal development when sympathetic axons are ramifying extensively in their targets in vivo. SCG neurons expressed all receptors capable of participating in GDF5 signaling at this stage of development. Using compartment cultures, we demonstrated that GDF5 exerted its growth promoting effect by acting directly on axons and by initiating retrograde canonical Smad signalling to the nucleus. GDF5 is synthesized in sympathetic targets, and examination of several anatomically circumscribed tissues in Gdf5 null mice revealed regional deficits in sympathetic innervation. There was a marked, highly significant reduction in the sympathetic innervation density of the iris, a smaller though significant reduction in the trachea, but no reduction in the submandibular salivary gland. There was no reduction in the number of neurons in the SCG. CONCLUSIONS: These findings show that GDF5 is a novel target-derived factor that promotes sympathetic axon growth and branching and makes a distinctive regional contribution to the establishment of sympathetic innervation, but unlike NGF, plays no role in regulating sympathetic neuron survival.

Leptin stimulates sympathetic axon outgrowth.

The neurohormone leptin regulates energy homeostasis. Circulating levels of leptin secreted by adipose tissue act on hypothalamic neurons in the brain leading to decreased appetite and increased energy expenditure. Although leptin signaling in the central nervous system (CNS) is fundamental to its ability to regulate the body's metabolic balance, leptin also has a variety of effects in many peripheral tissues including the heart, the liver, and the sympathetic nervous system. Leptin stimulation of the hypothalamus can stimulate glucose uptake via the sympathetic nervous system in heart, muscle, and brown adipose tissue. Leptin receptors (Ob-Rb) are also expressed by peripheral sympathetic neurons, but their functional role is not clear. In this study, we found that leptin stimulates axonal growth of both adult and neonatal sympathetic neurons in vitro. Leptin stimulates acute activation of the transcription factor STAT3 via phosphorylation of tyrosine 705. STAT3 phosphorylation is required for leptin-stimulated sympathetic axon outgrowth. Thus, circulating levels of leptin may enhance sympathetic nerve innervation of peripheral tissues.

Development of neuropeptide Y-containing neurons in sympathetic ganglia of rats.

Expression of neuropeptide Y (NPY) in the sympathetic ganglia was investigated by immunohistochemistry and tract tracing. The distribution of NPY immunoreactivity (IR) was studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 2-month-old, 6-month-old, 24-month-old). We observed that the percentage of NPY-IR neuronal profiles increased during early postnatal development. In the SCG and SG, the percentage of NPY-IR profiles enlarged in the first month of life from 43±3.6% (SCG) and 46±3.8% (SG) until 64±4.1% (SCG) and 58±3.5% (SG). The percentage of NPY-IR profiles in the CG increased during the period between 20days (65±3.8%) and 30days (82±5.1%) of animals' life and did not change in further development. In newborn and 10-day-old rats, a large portion of NPY-IR neurons was also calbindin D28K (CB)-IR in all sympathetic ganglia. The proportion of CB-IR substantially decreased during next 10days in the SCG, SG and CG. NPY-IR was approximately present in a half of the postganglionic neurons innervating muscle vessels of the neck and forearm, and the percentage of labeled NPY-IR profiles did not change during the development. Only single Ki67-IR neurons were also NPY-IR in the SCG, SG and CG in newborns and not in older animals. No NPY+/caspase 3+IR neurons were observed. Finally, the process of morphological changes in the size and percentages of NPY-IR profiles is complete in rats by the first month of life.

Hes1 is required for the development of the superior cervical ganglion of sympathetic trunk and the carotid body.

Hes1 gene represses the expression of proneural basic helix-loop-helix (bHLH) factor Mash1, which is essential for the differentiation of the sympathetic ganglia and carotid body glomus cells. The sympathetic ganglia, carotid body, and common carotid artery in Wnt1-Cre/R26R double transgenic mice were intensely labeled by X-gal staining, i.e., the neural crest origin. The deficiency of Hes1 caused severe hypoplasia of the superior cervical ganglion (SCG). At embryonic day (E) 17.5-E18.5, the volume of the SCG in Hes1 null mutants was reduced to 26.4% of the value in wild-type mice. In 4 of 30 cases (13.3%), the common carotid artery derived from the third arch artery was absent in the null mutants, and the carotid body was not formed. When the common carotid artery was retained, the organ grew in the wall of the third arch artery and glomus cell precursors were provided from the SCG in the null mutants as well as in wild-types. However, the volume of carotid body in the null mutants was only 52.5% of the value in wild-types at E17.5-E18.5. These results suggest that Hes1 plays a critical role in regulating the development of neural crest derivatives in the mouse cervical region.

Calbindin-D28k immunoreactivity in sympathetic ganglionic neurons during development.

Expression of CB in the sympathetic ganglia was investigated by immunohistochemistry. The distribution of CB immunoreactivity was studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from rats and cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, two-month-old, six-month-old). We observed that the percentage of CB-immunoreactive (IR) neurons decreased during early postnatal development in rats and cats. In all studied ganglia of both species, the percentage of CB-IR neurons was high in newborn and 10-day-old animals and significantly decreased up to 30 days of life. In rats of all ages, the largest percentage of CB-IR neurons was observed in the SG compared to the SCG and CG. In the cat sympathetic ganglia, the number of CB-IR neurons decreased more rapidly during the first two months of life, and only scattered CB-IR neurons were found in the sympathetic ganglia of two-month-old and six-month-old cats. In cats, the highest percentage of CB-IR neurons was observed in the SG, while the lowest percentage was found in the CG. The difference in size between CB+ and CB- neurons equally changed during development. Finally, the changes in the size and percentages of CB-IR neurons were complete in rats at the first month of life, and in cats at the end of the second month.

SCG postnatal remodelling--hypertrophy and neuron number stability--in Spix's yellow-toothed cavies (Galea spixii).

Whilst a fall in neuron numbers seems a common pattern during postnatal development, several authors have nonetheless reported an increase in neuron number, which may be associated with any one of a number of possible processes encapsulating either neurogenesis or late maturation and incomplete differentiation. Recent publications have thus added further fuel to the notion that a postnatal neurogenesis may indeed exist in sympathetic ganglia. In the light of these uncertainties surrounding the effects exerted by postnatal development on the number of superior cervical ganglion (SCG) neurons, we have used state-of-the-art design-based stereology to investigate the quantitative structure of SCG at four distinct timepoints after birth, viz., 1-3 days, 1 month, 12 months and 36 months. The main effects exerted by ageing on the SCG structure were: (i) a 77% increase in ganglion volume; (ii) stability in the total number of the whole population of SCG nerve cells (no change--either increase or decrease) during post-natal development; (iii) a higher proportion of uninucleate neurons to binucleate neurons only in newborn animals; (iv) a 130% increase in the volume of uninucleate cell bodies; and (v) the presence of BrdU positive neurons in animals at all ages. At the time of writing our results support the idea that neurogenesis takes place in the SCG of preás, albeit it warrants confirmation by further markers. We also hypothesise that a portfolio of other mechanisms: cell repair, maturation, differentiation and death may be equally intertwined and implicated in the numerical stability of SCG neurons during postnatal development.

Stargazin-related protein γ7 is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth.

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca²âº channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA-YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling.

[Age factors in the background electrical activity of the superior cervical ganglion neurons in rats].

Background discharges of single neurons were studied from the superior cervical ganglion in newborn, 10-, 20-day-old, 1-, 2- and 6-month-old rats. In all age groups, the largest proportion of neurons exhibited aperiodic activity. The percentage of neurons with respiratory rhythmic was less. In newborn and 10-day-old rats, the frequency of discharges was low. Discharge frequency increased in 20-day-old rats. In 20-day-old and more adult rats, we found neurons bursting with cardiac frequency. The means of frequency did not statistically differ in 1-, 2- and 6-month-old rats. Thus, the pattern of neuronal activity is formed during the development in 20-day-old rats. Final maturation of this pattern is observed in 1-month-old rats.

Stereological and allometric studies on neurons and axo-dendritic synapses in the superior cervical ganglia of rats, capybaras and horses.

The superior cervical ganglion (SCG) in mammals varies in structure according to developmental age, body size, gender, lateral asymmetry, the size and nuclear content of neurons and the complexity and synaptic coverage of their dendritic trees. In small and medium-sized mammals, neuron number and size increase from birth to adulthood and, in phylogenetic studies, vary with body size. However, recent studies on larger animals suggest that body weight does not, in general, accurately predict neuron number. We have applied design-based stereological tools at the light-microscopic level to assess the volumetric composition of ganglia and to estimate the numbers and sizes of neurons in SCGs from rats, capybaras and horses. Using transmission electron microscopy, we have obtained design-based estimates of the surface coverage of dendrites by postsynaptic apposition zones and model-based estimates of the numbers and sizes of synaptophysin-labelled axo-dendritic synaptic disks. Linear regression analysis of log-transformed data has been undertaken in order to establish the nature of the relationships between numbers and SCG volume (V(scg)). For SCGs (five per species), the allometric relationship for neuron number (N) is N=35,067xV (scg) (0.781) and that for synapses is N=20,095,000xV (scg) (1.328) , the former being a good predictor and the latter a poor predictor of synapse number. Our findings thus reveal the nature of SCG growth in terms of its main ingredients (neurons, neuropil, blood vessels) and show that larger mammals have SCG neurons exhibiting more complex arborizations and greater numbers of axo-dendritic synapses.

The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.

Maturation and long-term hypoxia alters Ca2+-induced Ca2+ release in sheep cerebrovascular sympathetic neurons.

The contribution of sympathetic nerves arising from the superior cervical ganglia (SCG) toward the growth and function of cerebral blood vessels is pertinent throughout maturation as well as in response to cardiovascular stress imposed by high-altitude long-term hypoxia (LTH). The function of SCG sympathetic neurons is dependent on intracellular Ca2+ concentration ([Ca2+]i) signaling, which is strongly influenced by a process known as Ca(2+)-induced Ca2+ release (CICR) from the smooth endoplasmic reticulum (SER). In this study, we used the sheep SCG neuronal model to test the hypotheses that maturation decreases CICR and high-altitude LTH depresses CICR in fetal SCG neurons but not in those of the adult. We found that the contribution of CICR to electric field stimulation (EFS)-evoked [Ca2+]i transients was greatest in SCG cells from normoxic fetuses and was abolished by LTH. The decline in CICR was associated with a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in fetal SCG cells during LTH, reducing SER Ca2+ levels below the threshold needed for the coupling of Ca2+ influx and CICR. With respect to the maturation from the fetus to adult, the decrease in CICR may reflect both a reduction in the levels of ryanodine receptor isoforms 2 and 3 and SERCA function. In response to LTH and in contrast to the fetus, CICR function in adult SCG cells is maintained and may reflect alterations in other mechanisms that modulate the CICR process. As CICR is instrumental in the function of sympathetic neurons within the cerebrovasculature, the loss of this signaling mechanism in the fetus may have consequences for the adaptation to LTH in terms of fetal susceptibility to vascular insults.

Synergistic effects of osteonectin and NGF in promoting survival and neurite outgrowth of superior cervical ganglion neurons.

Schwann cells (SCs) play a major role in the successful regeneration of peripheral nerves regeneration. Here we examined the effects of osteonectin (ON), a major factor secreted by SCs, on survival and neuritogenesis of mouse superior cervical ganglion (SCG) neurons. SC conditioned medium (SCCM) not only promoted the survival and neuritogenesis of SCG neurons at a level comparable to nerve growth factor (NGF) but also doubled the neurite length of NGF-treated SCG neurons. SCCM neuritogenic effects were not blocked by the tyrosine kinase receptor (Trk) inhibitor K252a demonstrating that these are not due solely to classical neurotrophic factors. Anti-ON neutralizing antibody diminished the SCCM-induced survival and neuritogenesis significantly. In the presence of K252a, the SCCM neuritogenic effects were blocked completely by anti-ON which suggests synergistic effects of ON with Trk-mediated growth factors. ON alone increased the survival and neurite outgrowth of SCG neurons significantly at high density cultures. ON at low concentration acts synergistically with NGF which induced maximum survival and neurite outgrowth (>50% increase). However, ON at high concentration was detrimental to survival (64% decrease) and neurite outgrowth (87% decrease) even in the presence of NGF. The well documented counter-adhesive effect of ON may account for this observation. Nevertheless, the growth promoting effects of ON became more pronounced as the cell density increased which suggests a possible interaction of ON with growth factors secreted by SCG neurons (autocrine or paracrine effects). Taken together, our study indicates that ON plays important roles in nervous system repair through its synergistic effects with growth factors.

The developing left superior cervical ganglion of Pacas (Agouti paca).

In this study the main question investigated was the number and size of both binucleate and mononucleate superior cervical ganglion (SCG) neurons and, whether post-natal development would affect these parameters. Twenty left SCGs from 20 male pacas were used. Four different ages were investigated, that is newborn (4 days), young (45 days), adult (2 years), and aged animals (7 years). By using design-based stereological methods, that is the Cavalieri principle and a physical disector combined with serial sectioning, the total volume of ganglion and total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal (somal) volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a 154% increase in the SCG volume, a 95% increase in the total number of mononucleate SCG neurons and a 50% increase in the total volume of SCG neurons. In conclusion, apart from neuron number, different adaptive mechanisms may coexist in the autonomic nervous system to guarantee a functional homeostasis during ageing, which is not always associated with neuron losses.

The developing and restructuring superior cervical ganglion of guinea pigs (Cavia porcellus var. albina).

Post-natal development comprises both maturation (from newborn to adult) and ageing (from adult to senility) and, during this phase, several adaptive mechanisms occur in sympathetic ganglia, albeit they are not fully understood. Therefore, the present study aimed at detecting whether post-natal development would exert any effect on the size and number of a guinea pig's superior cervical ganglion (SCG) neurons. Twenty right SCGs from male subjects were used at four ages, i.e. newborn (7 days), young (30 days), adult (7 months) and old animals (50 months). Using design-based stereological methods the volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a combination of post-natal-dependent increases and decreases in some variables: (i) 27% increase in ganglion volume, (ii) 24% and 43% decreases in the total number of mono and binucleate neurons, respectively, and (iii) 27.5% and 40% decreases in the mean perikaryal volume of mono and binucleate neurons, respectively. Despite the fall in neuron numbers found here, post-natal development is not only associated with neuron loss, but also embraces other structural adaptive mechanisms, which are discussed in this paper.

Asymmetric post-natal development of superior cervical ganglion of paca (Agouti paca).

Functional asymmetry has been reported in sympathetic ganglia. Although there are few studies reporting on body side-related morphoquantitative changes in sympathetic ganglion neurons, none of them have used design-based stereological methods to address this issue during post-natal development. We therefore aimed at detecting possible asymmetry-related effects on the quantitative structure of the superior cervical ganglion (SCG) from pacas during ageing, using very precise design-based stereological methods. Forty (twenty left and twenty right) SCG from twenty male pacas were studied at four different ages, i.e. newborn, young, adult and aged animals. By using design-based stereological methods the total volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated, using the vertical nucleator. The main findings of this study were: (1) the right SCG from aged pacas has more mononucleate and binucleate neurons than the left SCG in all other combinations of body side and animal age, showing the effect of the interaction between asymmetry (right side) and animal age, and (2) right SCG neurons (mono and binucleate) are bigger than the left SCG neurons (mono and binucleate), irrespective of the animal age. This shows, therefore, the exclusive effect of asymmetry (right side). At the time of writing there is still no conclusive explanation for some SCG quantitative changes exclusively assigned to asymmetry (right side) and those assigned to the interaction between asymmetry (right side) and senescence in pacas. We therefore suggest that forthcoming studies should focus on the functional consequences of SCG structural asymmetry during post-natal development. Another interesting investigation would be to examine the interaction between ganglia and their innervation targets using anterograde and retrograde neurotracers. Would differences in the size of target organs explain ganglia structural asymmetry?

Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration.

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration.

Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genes.

Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits alpha5, alpha7, or beta2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, alpha3, which is expressed in all SCG nAChRs, mRNA levels of beta4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, alpha4 declined sharply after birth and was barely detectable in adult animals. alpha5, alpha7, and beta2 subunit message levels also declined, though more slowly and less completely than alpha4. The subunits alpha6 and beta3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas alpha2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation.

Age-related structural and neurochemical changes of the human superior cervical ganglion.

The aim of this study was to investigate age-related morphological and neurochemical changes in the human superior cervical ganglion (SCG). Thirty-seven superior sympathetic human cervical ganglia of young, adult, and aged subjects were examined using morphometric analysis, biotin-streptavidin immunohistochemistry for detecting neurofilament, myelin protein, protein gene product 9.5, nerve growth factor receptor p75 in sympathetic neurons and nerve fibers. Morphometric parameters of neurons (area, long and short axis, shape factor of the neuron body, nucleus, cytoplasm, and lipofuscin) were investigated in every sixth serial section of the ganglion. Seven hundred neurons with clearly visible nuclei were measured in each studied group. The present study showed that human SCG of older subjects had larger areas of neuron body, cytoplasm and nucleus, a lower shape factor, an increased amount of lipofuscin, and a greater number of large-size neurons, as compared to SCG obtained from young subjects. Neuronal cytoskeletal alterations manifested themselves through a decreased number of neurofilament-positive neurons were detected in old human SCG. The amount of myelinated fibers decreased with age, although the amount of myelinated fibers in the young and the adult subjects varied from few to a moderate number. PGP 9.5 immunoreactivity varied in different age groups. A marked reduction of nerve growth factor receptor p75 in old human sympathetic neurons was detected. In conclusion, the findings of this study confirm age-related morphological changes in the human SCG. Structural neuronal changes may influence the deterioration of neuronal functional capacity, neuronal plasticity, and regenerative characteristics.