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In addition to their well-known actions within the nervous system, neurotrophins and their receptors are involved in immune system functioning, as demonstrated by their wide distribution in lymphoid tissues and their in vitro actions on immunocompetent cells. Nevertheless, the in vivo roles of neurotrophin-receptor systems in lymphoid tissues, as well as the scope of their influence throughout development and adulthood, are yet to be clarified. In the present study, we used combined morphological and immunohistochemical techniques to investigate the presence and cellular localization of p75NTR, the pan-neurotrophin receptor protein, in rat spleen from newborns to aging individuals, and the structural and innervation changes in the spleens of p75NTR-deficient mice. In rats, p75NTR was expressed by splenic nerve fibers and dendritic cells in an age-regulated fashion, with maximal expression detected at 2 weeks. Consistently, the spleens of newborn mice lacking this receptor protein showed no signs of ingrowing sympathetic fibers, along with an absence of defined white pulp areas. The present findings suggest a prolonged role of p75NTR in the physiology of the spleen; at least during the embryonic development period, the receptor may be critical for correct innervation and compartmentalization processes to occur., (Copyright 2003 Wiley-Liss, Inc.)

Glial cell line-derived neurotrophic factor promotes the survival of multiple neuron types in the central and peripheral nervous system. Moreover, it plays a key role in the development of the enteric nervous system and in the kidney organogenesis. Glial cell line-derived neurotrophic factor and their receptors are expressed in the developing tooth as well as in the trigeminal ganglion. However, the precise role of this growth factor in tooth morphogenesis and cell differentiation, or in the development of trigeminal ganglion cells, is still elusive. Using structural and ultrastructural techniques we analyzed in detail the first molar tooth germ of glial cell line-derived neurotrophic factor deficient mice as well as the neuronal density in trigeminal ganglion. The length and width of first molar tooth germ in knockout deficient animals showed no differences in the knockout animals in comparison with age-matched heterozygous or wild-type littermates. Nevertheless, in mice lacking glial cell line-derived neurotrophic factor, both ameloblasts and odontoblasts failed to fully develop and differentiate, and the enamel matrix and predentin layers were absent. On the other hand, the number of trigeminal sensory neurons and the structure of the nerves supplying first molar tooth germ were largely normal. Present results suggest a new non-neuronal role for glial cell line-derived neurotrophic factor in tooth development. Glial cell line-derived neurotrophic factor seems not to be involved in tooth initiation and morphogenesis, whereas it seems essential for cytodifferentiation. Conversely, neither development of trigeminal neuron nor nerve fibers supplying teeth are directly dependent on glial cell line-derived neutrophic factor.

The nerve growth factor (NGF) receptors p75LNGR and TrkA are expressed by thymic epithelial cells. Presumably, the NGF-TrkA system is involved in the paracrine communication between thymic epithelial cells and thymocytes, whereas the functional role of p75LNGR is still unknown. The thymus of vertebrates undergoes age-related changes that in part depend on hormonal factors. In order to find out whether thymic epithelial cells are responsive to NGF during the whole lifespan of the rat, we studied NGF receptor expression in the thymus from birth to 2 years of age, using immunohistochemistry. Furthermore, to evaluate whether increased plasma levels of NGF affected the ageing process, either NGF or 4-methylcatechol (4MC), an inductor of NGF synthesis, was administered. Both TrkA and p75LNGR were expressed by a subpopulation of thymic epithelial cells during the whole age range studied and their expression peaked at around 3 months. TrkA was primarily found in subcortical and medullary epithelial cells, whereas p75LNGR was seen in a subpopulation of medullary cells. Cortical epithelial cells, neural crest-derived cells, other stromal cells and thymocytes were not immunoreactive for NGF receptors. Neither the administration of NGF nor the increased NGF plasma levels obtained after 4MC treatment seemed to affect the ageing of the thymus as assessed by morphological and immunohistochemical criteria, but this increase in NGF levels did produce a shift in the expression of p75LNGR from epithelial cells to ED1-positive macrophages in animals of 6 months and older. Present results indicate that the expression of p75LNGR and TrkA in the rat thymus undergoes age-dependent changes that parallel those of epithelial cells. NGF could therefore be important for thymus homeostasis, possibly acting on epithelial cells. Nevertheless, NGF did not seem to be able to prevent the involution of this organ, although it produced a switch in the expression of p75LNGR, the significance of which remains to be established.

Neurocalcin (NC) is a recently characterized EF-hand calcium-binding protein present in a discrete population of sensory neurons and their peripheral mechanoreceptors, but its presence in peripheral nervous system neurons other than in the rat is still unknown. The present study was designed to investigate the occurrence of NC in the dorsal root ganglia (DRG) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, and rat), including humans. DRG were fixed, embedded in paraffin, and processed for immunohistochemistry using a polyclonal antibody against NC. The size of the immunoreactive neurons was measured. In all species examined, NC immunoreactivity (IR) was restricted to neurons but the percentage, as well as the size of the immunoreactive neurons, varied among different species. As a rule, small neurons (diameter <20 microm) lack NC IR. In some species (pig, dog, buffalo, cow), only the largest neurons showed IR, whereas in others (sheep, horse, rat, and humans) they covered the entire range of neuron sizes. The pattern of immunostaining was cytoplasmic, although in some species (cow and buffalo), it formed a peripheral "ring." The present results demonstrate that mammalian DRG contain a subpopulation of NC-positive neurons, which varies from one species to another. Based on the neuron size, the possible function of the NC-containing neurons is discussed., (Copyright 2000 Wiley-Liss, Inc.)

The development of Meissner-like and Pacinian corpuscles was studied in mice [from postnatal day (Pd) 0 to 42] by using immunohistochemistry for specific corpuscular constituents. The battery of antigens investigated included PGP 9.5 protein and neurofilaments, as markers for the central axon; S100 protein, vimentin, and p75(LNGFR) protein, to show Schwann-related cells; and epithelial membrane antigen to identify perineurial-related cells. In Meissner-like corpuscles immunoreactivity (IR) for neuronal markers was found by Pd7 and later. The lamellar cells of these corpuscles expressed first S100 protein IR (Pd7 to Pd42), then vimentin IR (Pd12 to Pd42), and transitory p75(LNGFR) IR (Pd7 to Pd19-20). Vimentin IR, but not epithelial membrane antigen, was detected in the capsule-like cells of the Meissner-like corpuscles. On the other hand, the density of Meissner-like corpuscles progressively increased from Pd0 to Pd19-20. Pacinian corpuscles were identified by Pd7. From this time to Pd42 the central axon showed IR for neuronal markers, and the inner core cells were immunoreactive for S100 protein. Moreover, vimentin IR was detected in the inner core cells by Pd19 and later. Unexpectedly, the central axons displayed S100 protein IR (from Pd7 to P28), while p75(LNGFR) protein IR or epithelial membrane antigen IR were never detected. Taken together, and based on the expression of the assessed antigens alone, the present results suggest that the Meissner-like and the Pacinian corpuscles in mice become mature around Pd19-Pd28 and Pd20, respectively. Furthermore, these results provide a baseline timetable for future studies in the normal or altered development of sensory corpuscles in mice since specific sensory corpuscles are functionally associated with different subtypes of sensory neurons the development of which is selectively disturbed in genetically manipulated mice., (Copyright 2000 Wiley-Liss, Inc.)

Increasing evidence suggests that some members of the family of the neurotrophins could be involved in immune system functioning. Both neurotrophins and their tyrosine-kinase signal-transducing receptors, the so-called Trk receptors, have been detected in various lymphoid tissues in a number of species. Nevertheless, their cellular localisation remains unclear in most cases. In this study, we used immunohistochemical techniques to localise TrkB in the rat spleen (from 0 days to 2 years). Cells expressing TrkB-like immunoreactivity were found exclusively within the white pulp of the spleen, along the marginal zone-follicle border and inside the follicles and periarteriolar lymphoid sheaths. These cells probably represented macrophage subpopulations, since they expressed the ED3 rat macrophage antigen. No evidence of TrkB-like protein expression in lymphocytes or follicular dendritic cells could be found. Furthermore, the density of TrkB-immunoreactive cells was observed to increase with age. Although the role of TrkB ligands in these cells remains to be clarified, the present findings provide further evidence for the supposed role of neurotrophins in immune system homeostasis.

Aim: This study analyses the occurrence and distribution of neurotrophins and their receptors in some types of tumours of neural-crest derived cells., Methods and Results: Light microscopy immunohistochemistry associated with quantitative image analysis was used to study the expression of neurotrophins (nerve growth factor, brain-derived neurotrophic factor and neurotrophin (NT)-3) and their cognate receptors (p75(LNGFR), TrkA, TrkB and TrkC) in histologically defined ganglioneuroma, phaeochromocytoma and paraganglioma. The material was fixed in 10% formaldehyde, paraffin-embedded and processed for indirect peroxidase immunohistochemistry using a battery of poly- and monoclonal antibodies to detect neurotrophins and their receptors, as well as some neuronal, endocrine and glial cell markers. A subpopulation of cells in phaeochromocytomas and ganglioneuromas expressed NT-3, but not other neurotrophins, while in paragangliomas no neurotrophins were detected. Regarding neurotrophin receptors, all tumours lacked p75(LNGFR), except for the ganglionic part of a case of mixed phaeochromocytoma, whereas they displayed TrkA (two of two ganglioneuromas, six of nine phaeochomocytomas and three of four paragangliomas). Furthermore, TrkC was regularly detected in a neuronal subpopulation in ganglioneuroma. Interestingly, the percentage of neurones expressing TrkA and TrkC was increased with respect to normal tissues in ganglioneuromas, as well as the percentage of the area occupied by TrkA-immunoreactive cells in the phaeochromocytomas., Conclusion: The pattern of expression of neurotrophins and neurotrophin receptors in the analysed tumours basically matches that of sympathetic neurones, adrenal chromaffin cells and paraganglionic cells, and suggests responsiveness of these cells to neurotrophins. Nevertheless, the function of TrkA and TrkC in regulating the biology of these tumours, if any, remains to be elucidated.

S100 protein in the vertebrate peripheral nervous system consists of homo- or heterodimers of S100alpha and S100beta proteins, the first predominating in neurons and the second in glial cells. Recently, however, occurrence of S100beta protein in neurons has been reported. The expression of S100 protein by Schwann cells, as well as their derivatives in sensory corpuscles, depends on the sensory axon (i.e., the Schwann cell-axon contact). The present study analyzed the distribution of S100alpha and S100beta proteins in human cutaneous sensory corpuscles and the effects of peripheral or central sensory axon severance in the expression of these proteins. Simple or double immunohistochemistry was carried out using a panel of antibodies against S100alpha, S100beta or S100alpha+beta proteins, and the sections were examined by light or laser confocal scanning microscopy. Skin samples were obtained from normal subjects and patients with spinal cord injury, nerve entrapment, and nerve sections plus graft. The lamellar cells of Meissner corpuscles as well as the inner-core lamellae of the Pacinian corpuscles displayed strong immunoreactivity (IR) for all antigens examined, the most intense labeling being obtained for S100beta protein. The pattern of immunostaining was unchanged after spinal cord injury, whereas the number of stained corpuscles as well as the intensity of IR for each antigen decreased in cutaneous sensory corpuscles after nerve injury, both entrapment and section plus graft. No evidence was found of axonal labeling. The present results provide evidence that Schwann-related cells in human cutaneous sensory corpuscles contain both S100alpha and S100beta and that the expression of these proteins is dependent on the functional and structural integrity of sensory fibers.

Human skin, including nerves and sensory corpuscles, displays immunoreactivity (IR) for low- (p75) and high-affinity (TrkA-like) receptors for nerve growth factor (NGF), the best characterized member of the family of neurotrophins. This study was designed to analyze the changes induced by spinal cord and peripheral nerve injuries in the expression of neurotrophin receptors in digital skin, with special reference to nerves and sensory corpuscles. Skin biopsy samples were obtained from 1) the hand and toes of normal subjects, 2) below the level of the lesion of patients with spinal cord injury affecting dorsal and lateral funiculi, 3) the cutaneous territory of entrapped peripheral nerves (median and ulnar nerves), and 4) the cutaneous territory of sectioned and grafted nerves (median nerve). The pieces were formalin-fixed and paraffin-embedded, cut in serial sections, and processed for immunohistochemistry using antibodies against human p75 and TrkA proteins. The percentage of sensory corpuscles displaying IR for p75 and TrkA-like, as well as the intensity of IR developed within them, was assessed using quantitative image analysis. Spinal cord severance causes a decrease in p75 IR in Meissner and Pacinian corpuscles, whereas TrkA-like IR did not vary. In other nonnervous tissues (i.e., epidermis, sweat glands), both p75 and TrkA-like IR was diminished or even absent. Similar but more severe changes were encountered in the skin from the territory of entrapped nerves. Finally, in subjects with sectioned-grafted nerves, p75 IR was found close to controls in nerves, reduced in Meissner corpuscles, and absent in the inner core of the Pacinian ones; TrkA-like IR was in the perineurium, a small percentage of Meissner corpuscles (about 7%), and the outer core and capsule of the Pacinan corpuscles. In the nonnervous tissues, p75 IR was practically absent, whereas TrkA-like IR did not change. No changes in the expression of neurotrophin receptors were observed in Merkel cells of the different groups. Present results show the following: 1) expression of nerve p75 IR in human cutaneous sensory corpuscles is sensitive to central deafferentation, to blockade or difficulty in axonal transport, and to disruption of axonal continuity independently of possible restoration of axonal integrity due to grafts; 2) expression of TrkA-like IR in nerves and sensory corpuscles is sensitive only to nerve transection; 3) the corpuscular Schwann-related cells are the only cells involved in the above modifications, the perineurial cells remaining unchanged; 4) the expression of p75 and TrkA-like IR by Merkel cells is independent of normal innervation; 5) an adequate innervation of the skin seems to be necessary for the expression of p75 but not TrkA-like in nonneuronal cells, especially in the epidermis. A role for NGF in the maintenance of epidermis integrity is discussed.

Increasing evidence suggests that, in addition to peripheral sensory and sympathetic neurons, the enteric neurons are also under the control of neurotrophins. Recently, neurotrophin receptors have been detected in the developing and adult mammalian enteric nervous system (ENS). Nevertheless, it remains to be established whether neurotrophin receptors are expressed in all enteric neurons and/or in glial cells and whether expression is a common feature in the enteric nervous system of all mammals or if interspecific differences exist. Rabbit polyclonal antibodies against Trk proteins (regarded as essential constituents of the high-affinity signal-transducing neurotrophin receptors) and p75 protein (considered as a low-affinity pan-neurotrophin receptor) were used to investigate the cell localization of these proteins in the ENS of adult man, horse, cow, sheep, pig, rabbit, and rat. Moreover, the percentage of neurons displaying immunoreactivity (IR) for each neurotrophin receptor protein was determined. TrkA-like IR and TrkC-like IR were observed in a neuronal subpopulation in both the myenteric and submucous plexuses, from esophagus to rectum in humans, and in the jejunum-ileum of the other species. Many neurons, and apparently all glial cells, in the human and rat enteric nervous system also displayed p75 IR. TrkB-like IR was found restricted to the glial cells of all species studied, with the exception of humans, in whom IR was mainly in glial cells and a small percentage of enteric neurons (about 5%). These findings indicate that the ENS of adult mammals express neuronal TrkA and TrkC, glial TrkB, and neuronal-glial p75, this pattern of distribution being similar in all examined species. Thus, influence of specific neurotrophins on their cognate receptors may be considered in the physiology and/or pathology of the adult ENS.

Increasing evidence suggests that some members of the neurotrophic factor family of neurotrophins could be implicated in the regulation of immune responses. Neurotrophins, as well as their tyrosine kinase signal-transducing receptors (the so-called Trk neurotrophin receptors), have been detected in different lymphoid tissues, although their cellular localization is not well known. In this study we used single and double immunohistochemistry to localize TrkB in situ in the rat thymus (in animals from 0 days to 2 years of age), in cytospin preparations of rat thymic cells, and in two mouse monocyte-macrophage cell lines (RAW 264.7 and J774A.1). We found TrkB protein expression in a subpopulation of cells in the corticomedullary junction, which simultaneously expressed the rat macrophage marker ED1. The density of TrkB-expressing cells increased with age, reaching maximal values at 2 years. Conversely, no evidence of TrkB protein expression could be found in dendritic cells, epithelial cells or thymocytes. Thymic macrophages in cytospin preparations, as well as in the mouse monocyte macrophage cell lines, also expressed TrkB protein. Although the possible function of TrkB in the thymic macrophage remains to be clarified, present findings add further evidence to the proposed role of neurotrophins in the immune system.

Pregnancy induces transient and reversible denervation of the mammalian uterus and uterine artery which origin remains still unclear. It is well established that the density of sympathetic innervation is regulated by the levels of peptidergic diffusible growth factors, especially nerve growth factor (NGF). Whether a decrease of NGF and/or its signal-transducing receptor TrkA are involved in this physiological denervation of the uterine artery during pregnancy has not been analyzed. The aim of the present study is to analyze this topic on human uterine artery using ELISA, Western blotting and immunohistochemistry (associated to quantitative image analysis). The material was obtained from surgical pieces (hysterectomy) of non-pregnant and pregnant women from 4 to 16 weeks of gestation. The density of innervation for tyrosine hydroxylase assessed in whole mount samples of uterine artery, as well as the density of nerve fibers identified with other general nerve (PGP 9.5 and NFP) or Schwann cell (S-100 protein) markers was significantly reduced (p<0.05) in the uterine artery from pregnant woman. On the other hand, the tissular levels of NGF, the density of TrkA, and the immunostaining for both NGF and TrkA, were significantly reduced in uterine arteries from pregnant patients. These results strongly suggest that the physiological denervation occurring in the uterine artery during pregnancy is related to a decrease in the availability of NGF by nerve fibers, and to the impossibility to mediate its effect due to a remarkable decrease in the signal-transducing TrkA receptor.

The occurrence of S100 proteins in neurons of the mammalian peripheral nervous system is still controversial. This study was designed to investigate this topic in dorsal root ganglia (DRG) and the enteric nervous system (ENS) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, rabbit and rat), as well as in DRG, paravertebral sympathetic ganglia (SG) and ENS of the adult man. Rat embryos of E17 and E19 were also examined. The material was fixed in Bouin's fixative, paraffin-embedded and processed for immunohistochemistry, combined with image analysis, using a panel of mono and polyclonal antibodies against S100alpha, S100beta or S100alpha + beta (referred to here as S100) proteins. In all species examined, strong S100 protein immunoreactivity (IR) was found in satellite glial cells and Schwann cells, which also showed S100alpha and S100beta IR in humans. Furthermore, faint S100 protein IR was observed in a subpopulation of DRG intermediate- and large-sized sensory neurons in humans, buffalo, sheep, and pig. The rat was the only species showing clear S100 and S100beta in neurons, labelling in about 30-35% in adults (small, intermediate and large in size), and about 88% at E17 and 42% at E19, respectively. Weak S100alpha protein IR was observed in most of human SG neurons. In ENS, S100 protein IR was restricted to enteric glial and Schwann cells, with the exception of cow and goat in which a subset of neurons in both the myenteric and submucous plexuses displayed strong S100 protein IR. Neuronal S100alpha IR and glial S100beta IR was found in the human ENS. The present results demonstrate intra- and inter-specific differences in the expression of S100 proteins by neurons of the peripheral nervous system among mammalian species. Furthermore, they also suggest that neuronal S100 protein, at least in humans, consists of both S100alpha and S100beta.

Background: Increasing evidence suggests that nerve growth factor (NGF), and probably other neurotrophins, are involved in the control of lymphoid organs and immunocompetent cells that express neurotrophins and/or their receptors. In the rat thymus, mRNA for TrkA (an essential component of the NGF signal transducing receptor) has been found primarily in stromal cells. The present study was undertaken to analyze the occurrence and localization of TrkA in the rat and human thymus, using Western blot and immunohistochemical techniques., Methods: Thymuses from human fetuses (estimated gestational ages of 29 and 32 weeks) and newborns (3 and 4 weeks old), as well as from 3-month-old rats were used. Human and rat samples were fixed in buffered 10% formaldehyde, paraffin-embedded, and processed for immunohistochemistry. Moreover, rat thymus samples were processed for Western blot analysis., Results: A protein band consistent with full-length TrkA (approximately 140 kDa) was detected in the rat thymus. Immunoreactivity (IR) for TrkA was exclusively found in thymic epithelial cells of both rat and human, identified because they also displayed cytokeratin IR. Interestingly, species-specific differences were noted for the expression of TrkA in different subtypes of thymic epithelial cells. Apparently, no immunolabelling was observed in other stromal cells or in lymphocytes., Conclusions: These results suggest that TrkA ligands may be involved in the control of thymic epithelial cells. This could be of potential importance because of the involvement of these cells in providing an appropriate microenvironment for maturation and selection of T lymphocytes.

Background: Trk proteins are essential constituents of the high-affinity signal-transducing neurotrophin receptors. They are expressed in a variety of non-neuronal tissues, including lymphoid organs, but their cellular localization in these remains to be established, as does the exact role of neurotrophins in the immune system. In this study we used immunohistochemical methods to analyze the cellular distribution of TrkA, TrkB, TrkC, and p75 (the low-affinity pan-neurotrophin receptor) proteins in normal human lymph nodes., Methods: Formaldehyde-fixed, paraffin-embedded human lymph nodes were processed for indirect immunoperoxidase labelling, using antibodies against each Trk protein, human p75, and a panel of antibodies against B-lymphocytes (CD20), macrophages (MAC387), dendritic cells (S-100 protein)., Results: Immunoreactivity (IR) for p75 was observed in follicular dendritic cells of lymphoid follicles, and possibly in B cells. TrkA-like IR was seen in dendritic cells and also in some follicular dendritic cells, and in blood vessel walls. TrKB-like IR labelled scattered cells, mostly in the T cell zones, identified as macrophages, while specific TrkC-like IR could not be observed in immunocompetent cells. In no case was Trk-like IR seen in lymphocytes., Conclusions: These results demonstrate the occurrence of Trk-like proteins in normal human lymph nodes and describe their cellular localization, favoring the notion that neurotrophins have a physiological role in the immune system, possibly acting through accessory cells and not directly on lymphocytes.

Background: The medial wall of the vomeronasal organ (VNO) is lined with a sensory epithelium that is closely related to the olfactory epithelium, which is developed from the olfactory placode. It undergoes continuous replacement during its life span. In other sensory epithelia, cell proliferation is under the control of some trophic factors. Whether these proteins are involved in the continuous turnover of the VNO epithelium is unknown. This study approaches this topic by analyzing the occurrence of signal-transducing receptor proteins for neurotrophins (Trk proteins) and epidermal growth factor (EGFr)., Methods: VNO samples were obtained from adult horses (n = 9) and processed for Western blot or immunohistochemical detection of TrkA, TrkB, TrkC, and EGFr. For immunohistochemistry, both frozen and formalin-fixed, paraffin-embedded sections were used. Antibodies against Trk proteins were polyclonal antibodies that map within the intracytoplasmic domain. Antibodies against EGFr were monoclonal antibodies that map within the external (clone EGFR1) or the cytoplasmic (clone F4) domains., Results: TrkA-like, but not TrkB- or TrkC-like, protein was detected in the VNO. By using immunoblotting, protein bands of TrkA-like protein with estimated molecular weights of 43-45, 55, and 60 kDa were found. In agreement with these findings, the sensory epithelium lining the VNO displayed strong TrkA-like immunoreactivity. On the other hand, regular protein bands with estimated molecular weights of 100 and 170 kDa, corresponding with immature and full-length EGFr, respectively, were found with the clone F4, whereas the clone EGFR1 was ineffective in detecting EGFr with Western blot analysis. Positive EGFr immunolabelling was observed regularly in the supranuclear pole of the sensory epithelial cells, and the pattern was identical with both antibodies used., Conclusions: The present results provide evidence for the occurrence of EGFr in the VNO of the adult horse, suggesting a role for their ligands (EGF and transforming growth factor-alpha) in this organ, probably in continuous cell replacement, during the adult life span. However, although immunoreactivity for TrkA-like protein was regularly observed, because the full-length protein was not found, whether or not its putative ligands (nerve growth factor and neurotrophin-3) act on these cells remains to be demonstrated.

This study was aimed at analyzing the changes in cutaneous sensory corpuscles from the territory of lesioned nerves and clinically denervated skin of patients with spinal cord injury using immunohistochemical methods. The morphological and biochemical characteristics of the Schwann-related cells of the mature sensory corpuscles (lamellar cells of Meissner corpuscles and inner-core of Pacinian corpuscles) depend upon the axon. To clarify whether this dependence requires structural and/or functional integrity of sensory axons we analyzed immunohistochemically some axonal, Schwann cell and perineurial cell antigens in cutaneous sensory corpuscles from i) the underlesional levels of patients with spinal cord injury affecting dorsal and lateral funiculi; ii) peripheral nerve entrapment, iii) sectioned and grafted nerves. Skin biopsy samples from the hand or feet were processed for peroxidase-antiperoxidase immunohistochemistry using monoclonal antibodies directed against neurofilament proteins (to label the axons), S-100 protein (to label Schwann-related cells), epithelial membrane antigen (to label the perineurial derivatives), vimentin (to label both Schwann cell and perineurial derivatives). Meissner and Pacinian corpuscles of subjects suffering from spinal cord lesions showed an immunohistochemical profile close to normality, although in many of them S-100 protein was unevenly distributed or absent. Sensory corpuscles of the cutaneous territory of entrapped nerves were in most cases similar to those of normally innervated skin. The most striking finding in these subjects was the hyperinnervation of blood vessels and sweat glands. Finally, nerve section and subsequent unsuccessful graft repair resulted in absence of immunostaining for all the assessed antigens in sensory corpuscles. The present results suggest that structural, but not functional, integrity of the axon is essential in maintaining some immunohistochemical characteristics of the human cutaneous sensory corpuscles. Morphological findings are correlated and discussed in relation to the clinical evaluation of the sensitivity.

Transforming growth factor-alpha (TGFalpha) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG. However, it remains to be elucidated whether all DRG neurons express EGFr or whether differences exist among neuronal subtypes. This study was undertaken to investigate these topics in adult human DRG using immunoblotting, and combined immunohistochemistry and image analysis techniques. A mouse monoclonal antibody (clone F4) mapping within the intracytoplasmic domain of EGFr was used. Immunoblotting revealed two main proteins with estimated molecular masses of approximately/equal to 65 kDa and 170 kDa, and thus consistent with the full-length EGFr. Additional protein bands were also encountered. Light immunohistochemistry revealed specific immunoreactivity (IR) for EGFr-like proteins in most (86%) primary sensory neurons, the intensity of immunostaining being stronger in the small- and intermediate-sized ones. Furthermore, EGFr-like IR was also observed in the satellite glial cells of the ganglia as well as in the intraganglionic and dorsal root Schwann cells. Taken together, our findings demonstrate that EGFr, and other related proteins containing the epitope labeled with the antibody F4, are responsible for the EGFr IR reported in DRG. Furthermore, we demonstrated heterogeneity in the expression of EGFr-like IR in adult human primary sensory neurons, which suggests different responsiveness to their ligands.

We investigated the expression of immunoreactivity (IR) for low- (p75) and high-affinity (trk proteins) neurotrophin-receptor proteins in adult human paravertebral-sympathetic ganglion neurons. Mouse monoclonal antibodies against the pan-neurotrophin-receptor p75, and rabbit polyclonal antibodies against specific epitopes of the intracytoplasmic domain on trk neurotrophin-receptor proteins were used in fresh unfixed and formaldehyde-fixed paraffin-embedded sympathetic ganglia. All adult human paravertebral-sympathetic neurons displayed trkA neurotrophin-receptor-like protein IR, 10% express trkC neurotrophin-receptor-like protein IR, 37-44% show p75 IR, and no IR was obtained for trkB neurotrophin-receptor-like protein. The intensity of immunostaining was independent of the neuron size. Labelling of non-neuronal tissues, especially blood-vessel walls, was observed for p75, trkA and trkC neurotrophin-receptor proteins. These results indicate that overlapping exists in the expression of p75 and trk neurotrophin-receptor proteins in adult human paravertebral-sympathetic neurons, and suggest that neurotrophins might act on these neurons.

Background: The cytoskeleton of mature neurons consists of three main types of filamentous structures: microtubules (or neurotubules) neurofilaments and microfilaments, and of the so-called associated proteins. Neurotubules are formed by alpha- and beta-tubulin; neurofilaments are comprised of three protein subunits (68, 160, and 200 kDa of molecular weight), referred to here as neurofilament proteins (NFPs). The microtubule-associated proteins (MAPs) and tau-proteins form cross bridges between microtubules and other cytoskeletal constituents, as well as cellular organelles. This study analyzes the distribution of several cytoskeletal proteins in adult human dorsal root ganglia (DRG)., Methods: Sections of formaldehyde-fixed, paraffin-embedded adult human DRG were processed for PAP immunohistochemistry. Mouse monoclonal antibodies against specific epitopes of alpha- and beta-tubulin, MAP-1, MAP-2, MAP-5, tau-protein, and NFPs (68, 160, and 200 kDa) were used. Furthermore, a quantitative image analysis (optic microdensitometry) was performed to establish the relationship between neuronal size and intensity of immunostaining., Results: Most of DRG neuron cell bodies displayed immunoreactivity for all assessed antibodies, with the exception of MAP2, which was absent. Nevertheless, the neuronal perikarya showed an heterogeneous pattern of immunoreactivity, which was not related to neuronal profile size. Positive immunolabelling was also observed in satellite cells and Schwann cells for microtubule and MAP1 proteins, and for tau-protein in Schwann cells., Conclusions: Adult human primary sensory neurons in DRG express immunoreactivity for neurotubule and neurofilament proteins, as well as for some microtubule-associated proteins. However, since large heterogeneity was observed in the expression of those proteins, we conclude that the expression of cytoskeletal proteins is not a criterion to establish DRG neuronal subtypes.

Herbst corpuscles are the avian equivalent to the mammalian Pacinian corpuscles. In this study we used indirect peroxidase-anti peroxidase (PAP) immunohistochemistry to analyze the distribution in the pigeon cutaneous Herbst corpuscles, of several markers which are known to specifically label the axon, the Schwann-related and perineurial-related cells in Pacinian corpuscles. The distribution of the assessed antigens on Herbst corpuscles was as follows: i) the central axon displayed positive immunoreactivity for neurofilament proteins and neuron specific enolase; ii) the lamellar cells forming the inner-core were positive for S-100 protein and vimentin, whereas fibroblast surrounding them were vimentin and epithelial membrane antigen positive; iii) the capsule was focally immunolabelled for vimentin, and regularly for epithelial membrane antigen. No immunoreactivity was found neither for cytokeratins nor for glial fibrillary acidic protein. These observations demonstrate that the immunohistochemical profile of cutaneous (beak skin and rictus) Herbst corpuscles in pigeon is similar to that of the mammalian Pacinian corpuscles.

The occurrence and distribution of beta-amyloid precursor protein (beta APP) and of beta-amyloid peptide (beta/A4) was investigated using immunoblotting and immunohistochemical techniques in the digital skin of healthy adult subjects. beta APP-like proteic bands with apparent molecular masses between 55-60 kDa, 100-125 kDa (corresponding to the full-length beta APP isoforms), 145-150 kDa, and 200 kDa were found in pellets and supernatants of whole skin and dermis. The same proteins, except that of approximately 200 kDa, were also found in pellets from the epidermis, whereas epidermic supernatants were unreactive. beta/A4 was not found by immunoblotting. Light microscope immunohistochemistry showed beta APP immunoreactivity (IR) in: (a) dermal nerves; (b) lamellar cells of Meissner, as well as inner-core, outer-core and capsule of Pacinian corpuscles; and (c) dermal blood vessels, sweat glands and, occasionally, epidermis. The distribution of beta/A4 IR matched that of beta APP, and no evidence of extracellular beta/A4 IR was encountered. Present results demonstrate that beta APP, but not beta/A4, is normally present in human glabrous (digital) skin. The potential clinical relevance of these findings is discussed.

The influence of long term treatment with the dihydropyridine-type Ca2+ antagonist darodipine (PY 108-068) on age-dependent changes in calbindin D-28K immunoreactivity in the cerebellar cortex of male Wistar rats was assessed. In 12-month-old rats used as an adult reference group, specific calbindin D-28K immunoreactivity was found within the cytoplasm of Purkinje neurons and their dendritic processes. The number of Purkinje neurons displaying calbindin D-28K immunoreactivity was decreased in the cerebellar cortex of aged in comparison with adult rats. The pattern of calbindin D-28K immunoreactivity was similar in the cerebellar cortex of 24-month-old rats (aged), although a significant decrease in the intensity of immunoreactivity was noticeable. Treatment of aged rats with darodipine for 6 months increased the percentage of immunoreactive Purkinje neurons and the intensity of calbindin D-28K immunoreactivity in the cytoplasm of Purkinje neurons. Calbindin D-28K is a Ca2+ binding protein probably involved in the modulation of Ca2+ homeostasis. The observation of a positive effect of darodipine treatment on calbindin D-28K immunoreactivity in the cerebellar cortex suggests that manipulation of dihydropyridine-type Ca2+ channels may contribute to counter age-dependent changes of Ca2+ homeostasis.

The influence of aging and of treatment with the dihydropyridine Ca2+ antagonist darodipine (PY 108-068) on the age-related microanatomical changes of rat brain were studied in male Wistar rats treated from the 18th to the 24th month of age with an oral dose of 5 mg/kg/day of darodipine. Twelve-month-old untreated rats were used as an adult reference group. A decreased number of nerve cells and of alkaline phosphatase-positive capillaries and an increased lipofuscin deposition were observed in the frontal and occipital cortex, in the hippocampus, and in the cerebellar cortex of rats of 24 months in comparison with 12-month-old animals. The number of nerve cells was higher in the occipital cortex and in the hippocampus, but not in the frontal cortex and in the cerebellar cortex, of darodipine-treated rats in comparison with age-matched untreated animals. Lipofuscin deposition is reduced in all the brain areas investigated. The density of alkaline phosphatase-reactive capillaries is also increased in the frontal and occipital cortex and in the hippocampus of aged rats treated with darodipine. The above results suggest that treatment with darodipine is able to counter some microanatomical changes occurring in the brain of aged rats and involving not only microvascular parameters. The occipital (visual) cortex and the hippocampus were the cerebral areas more sensitive to treatment with darodipine. The possible relevance of these findings is discussed.

The distribution of the beta-amyloid precursor protein (betaAPP) in the human gastrointestinal tract, from esophagus trough rectum, was studied using immunoblotting, as well as combined immunohistochemical and image analysis (optic microdensitometry) techniques. The study was focused on the enteric nervous system. betaAPP was detected by means of a monoclonal antibody (22C11), which recognizes all betaAPP isoforms as well as betaAPP-like proteins. Immunoblotting revealed two main protein bands, one corresponding to full-length betaAPPs (estimated molecular masses of approximately 97-115 kDa); the other corresponded to a protein with estimated molecular masses of 55 kDa. Specific betaAPP immunoreactivity (IR) was found in the submucous and myenteric plexuses localized in the supporting glial cells rather than in neurons. Differences were encountered neither in the localization nor in the intensity of immunostaining among different segments of the gastrointestinal tract. Moreover, no age-dependent changes were found. betaAPP IR was also regularly observed in blood vessels, primarily labelling endothelial cells. Our results provide evidence for the occurrence of betaAPP in human gastrointestinal tract of healthy people in both neuronal and nonneuronal tissues. Whether or not these findings have functional or clinical relevance remains to be clarified in future studies.

We used immunohistochemical techniques and monoclonal antibodies to localize two basement membrane components (laminin and type IV collagen) in the nerves and sensory nerve formations, or corpuscles, supplying human digital skin. Furthermore, neurofilament proteins, S-100 protein and epithelial membrane antigen were studied in parallel. In dermal nerve trunks, immunostaining for laminin and type IV collagen was found to be co-localized in the perineurium and the Schwann cells, the stronger immunoreactivity being at the external surface of the cells. In the Meissner digital corpuscles, the immunoreactivity for laminin and type IV collagen was mainly observed underlying the cell surface of lamellar cells, while the cytoplasm was weakly immunolabelled or unlabelled. Finally, within Pacinian corpuscles co-localization of the two basement membrane molecules was encountered in the inner core, intermediate layer, outer core and capsule. Laminin and type IV collagen immunoreactivities were also found in blood vessels and sweat glands, apparently labelling basement membrane structures. The present results provide evidence for the presence of basement membrane in all periaxonic cells forming human cutaneous sensory nerve formations, and suggest that all of them are able to synthesize and release some basement membrane components, such as laminin and type IV collagen. The possible role of laminin in sensory nerve formations is discussed.

The distribution of neurotrophin receptors (p75, trkA-, trkB-, and trkC-receptor proteins) was studied by immunohistochemistry on sections of human gastrointestinal tract mucosa from esophagus through rectum. Moreover, chromogranin A (CgA) was studied in parallel to identify endocrine cells (EC). In all of the analyzed samples there was specific immunoreactivity (IR) for trkB-receptor protein in EC, the percentage of which varied between 26 +/- 0.6% for the duodenum and 78 +/- 3% for the sigmoid colon. EC displaying trkC-receptor protein IR were also encountered, in some cases, in EC of the gastric fundus (9%), duodenum (12%), jejune (23%), and colon (12%); trkA-receptor protein IR was occasionally present labelling EC in the jejune (52%), ileum (25%), and sigmoid colon (18%); finally, p75 was in 21% of EC exclusively in one case in the ileum. In addition to EC, IR for all assessed antigens was also present in the submucous blood vessels. Our results provide evidence for the occurrence of neurotrophin receptor proteins in nonneuronal tissues and suggest that neurotrophins, especially that binding trkB receptor proteins, can regulate a subpopulation of EC cells. However, whether EC expressing different trk receptor proteins represent neurochemical subtypes of EC, and whether the identified trk receptor proteins correspond to functional receptors, remain to be elucidated.

Background: Nerve growth factor (NGF) is produced in target tissues of sympathetic and neural-crest derived sensory neurons, including skin, to provide them trophic support. The biological effects of NGF on responsive cells are mediated by specific high-affinity receptors. Recently, a protein tyrosine kinase of congruent to 140 kDa molecular weight, encoded by the proto-oncogene trkA, has been identified as the high-affinity NGF receptor (gp140-trkA). The present work was undertaken to study the localization of gp140-trkA-like immunoreactivity (IR) in human peripheral ganglia (sympathetic and dorsal root ganglia), and in glabrous skin., Methods: Lumbar dorsal root ganglia, para- and prevertebral sympathetic ganglia, and digital glabrous skin were studied immunohistochemically using a rabbit anti-gp140-trkA polyclonal antibody. In order to accurately establish the localization of gp140-trkA IR, the neurofilament proteins and S-100 protein were studied in parallel in: (1) sensory and sympathetic ganglia, to label neuron cell bodies and satellite or supporting cells, respectively; (2) human skin, to label axons, Schwann and related cells within nerves and sensory corpuscles. Moreover, a quantitative study (neuron size, intensity of immunostaining) was carried out on sympathetic and dorsal root ganglia neuron cell bodies., Results: A specific gp140-trkA-like IR was found in: (1) a subpopulation (65%) of primary sensory neuron cell bodies, including most of the large-sized ones but also small- and intermediate-sized ones; (2) most of sympathetic neuron cell bodies (82%); (3) the perineurial cell, Schwann cells, and large axons of the nerve trunks supplying digital skin; (4) the lamellar cells of Meissner corpuscles; (5) the central axon, inner-core, outer-core, and capsule of Pacinian corpuscles. In addition, the occurrence of gp140-trkA-like IR was observed in some non-nervous tissues of the skin, including epidermis (mainly in the basal layer), sweat glands, and arterial blood vessels., Conclusions: Present results provide evidence for the localization of gp140-trkA-like IR in: (1) nerve cells which are known to be NGF-responsive, and (2) non-nervous cutaneous tissues which are innervated by NGF-dependent peripheral neurons. These findings suggest that, in addition to the well-established role of NGF on sensory and sympathetic neurons, this neurotrophin may be able to regulate some other functions on non-nervous cells which are targets for NGF-dependent peripheral neurons.

The present study reports the occurrence and localization of beta-amyloid precursor protein (APP) immunoreactivity (IR) in human lumbar dorsal root ganglia of healthy adult subjects (age range 25-43 years). To ascertain that ganglionic cells displayed APP IR, neurofilament (NFP) and S-100 proteins (S100P) were studied in parallel. Immunoblotting revealed four or five major proteins with apparent molecular masses between 100-125 kDa, which corresponded with the different full-length APP isoforms. Moreover, an additional protein of approximately 55 kDa was detected. Selective APP IR was observed restricted to the satellite glial cell cytoplasms whereas neuron cell bodies resulted unlabeled. Moreover, some intraganglionic nerve fibers also displayed APP IR, apparently labelling Schwann cells. No individual differences among subjects were observed neither in the pattern of APP IR distribution, nor in the intensity of APP IR. Although it remains to be demonstrated whether or not human primary sensory neurons express APP, present results strongly suggest that supporting glial cells may be a primary source of APP or any related peptide, at least in adult healthy people. The functional and clinical relevance of these findings, if any, remain to be clarified.

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.

The localization of the beta/A4 amyloid precursor protein (APP) was studied in the human lumbar paravertebral sympathetic ganglia of subjects of different ages, free of neurologic disease, using combined immunohistochemistry and image analysis techniques (optic microdensitometry). To ascertain which cells displayed APP-like immunoreactivity (APP-LI), S-100 and neurofilament proteins were studied in parallel to label the supporting glial cells and the neuron perikarya, respectively. Specific APP-LI was observed labelling both neuron cell bodies and supporting glial cells independently of age. In all cases, the intensity of immunostaining was stronger in glial cells than in neurons. Moreover, the intensity of APP-LI was independent of both age and neuron size. Present results provide evidence for the presence of APP-LI in the human sympathetic ganglia, and for the absence of changes in the expression of this protein, or proteins, with aging. The functional and clinical relevance of these findings remains to be clarified.

The rat dorsal root ganglia (DRG) contain heterogeneous subpopulations of sensory neurons as demonstrated by ultrastructural, histochemical and immunohistochemical methods. In this study we investigated whether phenotypic heterogeneity occurs in the distribution of protein gene product 9.5 (PGP 9.5) in DRG neurons of adult rats by combined immunohistochemical and image analysis (neuron-size and intensity of immunostaining) techniques. Moreover, the effect of different fixatives on the expression of PGP 9.5 was analyzed. PGP 9.5 immunoreactivity (IR) was observed in all primary sensory neurons and in the axons of the ganglionic nerve fibres, but not in the satellite glial cells or Schwann cells. Data from a quantitative study demonstrated that DRG neurons displayed a homogeneous pattern of PGP 9.5 IR which was not affected by fixatives, and no correlation between neuron size and intensity of immunostaining was encountered. Thus, as reported for other neuronal and neuroendocrine cell proteins, no heterogeneity exists in the phenotypic expression of immunohistochemically demonstrable PGP 9.5 in sensory neurons of the adult rat DRG.

Background: The epidermal growth factor receptor (EGFr) binds both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha), which are currently considered among putative growth factors playing a role in the nervous system. EGFr and their ligands have been localized in the mammalian peripheral nervous system. The present study was undertaken to investigate whether nerves and sensory corpuscles supplying human glabrous skin express EGFr., Methods: Formaldehyde fixed, paraffin embedded samples of finger-tip digital skin obtained from adult healthy subjects were processed for indirect PAP immunohistochemistry using a monoclonal antibody against an epitope of the intracellular domain of EGFr. To ascertain the localization of EGFr immunoreactivity, neurofilament proteins (NFP), S100 protein (S100P), and epithelial membrane antigen (EMA) were studied in parallel to label axons, Schwann cells, and perineurial cells, respectively, as well as their corpuscular derivatives., Results: A variable intensity of EGFr immunostaining was regularly observed in the perineurium and Schwann cells, and occasionally in the axons of nerve bundles. EGFr immunoreactivity was also present in the axon and lamellar cells of Meissner corpuscles, and within the axon, inner-core, outer-core, and capsule of Pacinian corpuscles., Conclusions: Present results demonstrate that human cutaneous nerves and sensory corpuscles express EGFr suggesting a role for peptides able to bind EGFr, i.e., EGF and TGF alpha, in the human peripheral nervous sensory system.

The distribution of several markers for Schwann cells and perineurial cells, including S-100 protein, Leu-7 antigen, vimentin and epithelial membrane antigen was studied immunohistochemically in Pacinian corpuscles from human digital skin. Formaldehyde fixed paraffin embedded tissues, and monoclonal antibodies were used. A positive immunostaining for S-100 protein, Leu-7 antigen and vimentin was found in the inner-core cells, whereas the outer-core and capsule lamellae were labelled for both epithelial membrane antigen and vimentin. The pattern of vimentin immunoreactivity was not homogeneous but focally distributed, and occasionally, positive immunoreactivity for vimentin was observed in blood vessels supplying the capsule. Present results provide evidence that some antibodies against Schwann cells and perineurial fibroblastic cells, selectively stain the Pacinian corpuscles derivatives while others are commonly expressed for non-neuronal cells of sensory nerve corpuscles.