Micro-structural geometry of thin films intended for the inner lumen of nerve conduits affects nerve repair.

Damage to peripheral nerves can cause significant motor or sensory injuries. In serious cases, a nerve is sacrificed from another part of the body to repair a damaged nerve (autograft). The development of biodegradable polymer conduits may offer an alternative to autografts. This study investigated the surface topography and mechanical properties of smooth, pitted and grooved structures of ultra-thin poly (ε-caprolactone)/poly lactic acid blended, solvent-cast films. We have investigated the effect of the groove shape on cell morphology and alignment. Photolithography and dry/wet etching was used to develop patterned silicon substrates with grooves with accurate geometries (V shaped, sloped walls and square shaped). Using a neural cell line (NG108-15), in vitro experiments confirmed good cell attachment and proliferation on all the polymer scaffolds. Imaging techniques demonstrated that there was different cellular responses and morphology according to the shape of the groove. Studies showed that the geometry, particularly the angle of the slope and the space between grooves, affected cellular responses. In addition, biomechanical studies showed that the patterned films had excellent mechanical properties and were stronger than the natural nerve. The conduit tubes were made by rolling the films around a mandrel and using a thermal welding technique to join the edges. The promising biomechanical and in vitro results demonstrate that nerve cell responses are affected by the shape of longitudinal grooves, and particularly by the angle of the slope of the groove walls.

The role of endogenous nerve growth factor in human diabetic neuropathy.

Nerve growth factor (NGF) is trophic to sensory and sympathetic fibers. In animal models, NGF is depleted in diabetic nerves and NGF deprivation produces hypoalgesia. Exogenous NGF can reverse some of the pathological changes in diabetic nerves and NGF excess leads to hyperalgesia. We have quantified sensory and autonomic function in early diabetic polyneuropathy and correlated changes with levels of NGF and neuropeptides in affected skin. We describe an early length-dependent dysfunction of sensory small-diameter fibers, prior to dysfunction of sympathetic fibers, with depletion of skin NGF and the sensory neuropeptide substance P. We describe a significant correlation between NGF depletion and decreased skin axon-reflex vasodilation, mediated by small sensory fibers partly via substance P release. Immunostaining shows depletion of NGF in keratinocytes in diabetic skin. We propose that a decrease in endogenous skin-derived NGF influences the presentation of diabetic polyneuropathy, although metabolic or vascular abnormalities may be the cause of the neuropathy. As loss of nociception and axon-reflex vasodilation contribute to diabetic foot ulceration, early and prolonged NGF treatment at an appropriate dose may provide rational prophylaxis for this condition.

Laryngeal transplantation in minipigs: vascular, myologic and functional outcomes.

There is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. Regenerative medicine offers promise, but cannot presently deliver implants with functioning neuromuscular units. A single well-documented laryngeal transplant in man was a qualified success, but more information is required before clinical trials may be proposed. We studied the early response of the larynx to laryngeal transplantation between 17 pairs of NIH minipigs full matched at the MHC2 locus. Following iterative technical improvements, pigs had good swallowing and a patent airway at 1 week. No significant changes in mucosal blood flux were observed compared with pre-operative measurements. Changes in muscle morphology and fibre phenotype were observed in transplant muscles retrieved after 7 days: the levels of fast and slow myosin heavy chain (MyHC) protein were reduced and embryonic MyHC was up regulated consistent with denervation induced atrophy. At 1 week laryngeal transplantation can result in good swallowing, and is not associated with clinical evidence of ischemia-reperfusion injury in MHC-matched pigs.

Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b.

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 micron, controls 70.8 micron; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types. This study indicates that NT-3-enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro-muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.

[Fibrin matrix enhances adherence of peripheral nerve regenerative cells]. / Fibrinmatrix erhöht adhärenz von regenerativen peripheren nervenzellen.

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Analysis of the dose-response of N-acetylcysteine in the prevention of sensory neuronal loss after peripheral nerve injury.

BACKGROUND: N-Acetylcysteine (NAC) is a safe pharmaceutical agent known to protect cells from oxidative damage. Following peripheral nerve transection, NAC has been found to eliminate sensory neuronal loss. This study examines the dose-response relationship of NAC in preventing neuronal death. METHODS AND FINDINGS: The rat sciatic nerve transection model was used, and stereological quantification of sensory neuron survival carried out at two weeks post-axotomy. NAC was administered systemically as an intraperitoneal injection to five groups of rats at a range of doses (1-300 mg/kg/day). Significant neuronal loss was observed in the 1 mg/kg/day dosage group (18.5% loss, p = 0.067 vs. sham treatment). A degree of neuroprotection occurred with 10 mg/kg/day (9.1% loss, p < 0.005 vs. control), whilst there was no significant loss with either 150 or 300 mg/kg/day. CONCLUSIONS: The prevention of sensory neuronal loss with NAC is dose dependent and effective over a wide therapeutic range. This analysis confirms the efficacy of systemic administration and provides a dose framework with which NAC has clinical potential to improve outcome after peripheral nerve trauma.

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a ß1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that ß1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

The biomechanical and functional relationships of the proximal radioulnar joint, distal radioulnar joint, and interosseous ligament.

This biomechanical study assessed integrated function of the proximal radioulnar joint (PRUJ), interosseous ligament (IOL), and distal radioulnar joint (DRUJ). Tekscan™ pressure sensors were inserted into the DRUJ and PRUJ of 15 cadaveric specimens. MicroStrain(®) sensors were mounted onto the IOL on nine of these specimens. A customized biomechanical jig was used to apply axial loads and take measurements through pronosupination. The PRUJ, IOL, and DRUJ were shown to function as an integrated osseoligamentous system distributing applied load. The PRUJ has transmitted pressure profiles similar to those of the DRUJ. Different IOL components support loading at different stages of pronosupination. The IOL is lax during pronation. Mid-IOL tension peaks in the midrange of forearm rotation; distal-IOL tension peaks in supination. Axial loading consistently increases IOL strain in a non-linear fashion. There are clinical implications of this work: disease or surgical modification of any of these structures may compromise normal biomechanics and function.

Endothelin-1 in human skin: immunolocalization, receptor binding, mRNA expression, and effects on cutaneous microvascular endothelial cells.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the maintenance of systemic and peripheral vascular tone. We have therefore sought direct evidence of a role for ET-1 in the regulation of blood flow and vascular tone in the human cutaneous microvasculature. Immunostaining for ET-1 was observed in all cutaneous blood vessels of normal human skin including the capillaries of the dermal papillae. Autoradiography showed specific binding of 125I-ET-1 over capillaries and larger blood vessels as well as hair follicles and sweat glands. In situ hybridization with a 32P-labeled RNA probe for ET-1 demonstrated mRNA for ET-1 in cultured human dermal microvascular endothelial cells (HDMEC). In HDMEC, basal release of PGE2 was significantly attenuated by ET-1 (100 pM-100 nM) (p less than 0.05, n = 7) with maximum inhibition in cells incubated with 10 nM ET-1. ET-1 also increased intracellular cAMP in a dose-dependent manner with a significant increase in HDMEC incubated with 100 nM ET-1 (p less than 0.05, n = 4). In HDMEC incubated with 100 nM ET-1, inhibition of PGE2 release was unaffected by the dihydropyridine Ca++ channel antagonist nifedipine or the extracellular Ca++ chelator EGTA, whereas the intracellular Ca++ chelator TMB-8 partially blocked the action of ET-1. In contrast, cAMP accumulation was significantly attenuated by EGTA (p less than 0.05, n = 4), nifedipine (p less than 0.05, n = 4), and TMB-8 (p less than 0.05, n = 4), indicating that the endothelial cell responses to ET-1 are complex and appear to involve both Ca(++)-sensitive and -insensitive pathways. These results provide evidence of an autocrine/paracrine role for ET-1 in the human cutaneous microvasculature.

Characterization of endothelin-binding sites in human skin and their regulation in primary Raynaud's phenomenon and systemic sclerosis.

Endothelin (ET), which mediates vasoconstrictor and vasodilator activities via multiple receptor subtypes, has been implicated in the control of blood flow and vascular tone in human skin, and possibly in the abnormal vasoconstrictor response in primary Raynaud's phenomenon and systemic sclerosis. Using in vitro autoradiography we have examined the endothelin-binding characteristics and receptor subtypes of human skin, and sought to provide evidence for endothelin receptor regulation in skin from patients with primary or secondary Raynaud's phenomenon. Specific 125I-ET-1 and 125I-ET-3 binding sites were localized to microvessels of the sub-epidermal plexus and dermal papillae, larger blood vessels, sweat glands, epidermis, and hair follicles. Both ETA and ETB receptors were demonstrated in microvessels and other structures. ET receptor heterogeneity in skin vasculature suggests a role for ET as an autocrine/paracrine regulator of vasoconstrictor and vasodilator pathways in human skin. The presence of binding sites in epidermis and hair follicles suggests a possible mitogenic function for endothelin in human skin. Endothelin-binding density was significantly higher (p < 0.05) in microvessels of skin from patients with systemic sclerosis but not significantly different in Raynaud's phenomenon patients, compared to controls. Lack of down regulation of ET receptors in Raynaud's phenomenon and systemic sclerosis may contribute to the pathogenesis of vasospasm in these diseases.

Neurotensin-like immunoreactivity in a subpopulation of noradrenaline-containing cells of the cat adrenal gland.

RIA of cat adrenal tissue extracts revealed a neurotensin-like immunoreactive material concentrated within the medulla of the gland (mean +/- SEM neurotensin concentration, 15.2 +/- 3.6 pmol/g whole gland; 47.9 +/- 18.4 pmol/g microdissected medulla). This immunoreactive material was found to elute in the region of synthetic neurotensin, thus indicating a similarity to the tridecapeptide originally isolated from bovine hypothalamus. Using immunocytochemical procedures at both light and ultrastructural levels, a neurotensin-like immunoreactive material was localized to a subpopulation of noradrenaline-containing cells quite distinct from the previously described enkephalin-immunoreactive chromaffin cells. Correlative ultrastructural observations have identified three morphologically distinct types of chromaffin cells in the medulla, indicating a marked heterogeneity within the noradrenaline cell population. The finding of neurotensin-like immunoreactivity in noradrenaline-containing cells of the cat adrenal medulla provides further evidence in support of the postulated existence of heterogeneous subpopulations of noradrenaline-containing cells and suggests a possible functional interrelationship between neurotensin and catecholamine.

Neuropeptide tyrosine (NPY) immunoreactivity in norepinephrine-containing cells and nerves of the mammalian adrenal gland.

The application of immunocytochemistry at both light and electron microscopic levels has revealed neuropeptide tyrosine (NPY)-immunoreactive material to be localized to norepinephrine-containing endocrine cells in the adrenal medulla and also to varicose nerve fibers penetrating the adrenal cortex of several mammalian species, including horse, cat, rat, guinea pig and mouse. Correlative electron microscopic immunostaining has revealed that enkephalin and NPY immunoreactivities are co-localized to the same norepinephrine-containing secretory granules. High concentrations of NPY have been extracted from the mouse adrenal gland (1243.7 +/- 122.8 pmol NPY/g wet tissue; mean +/- SE). Chromatographic analysis has shown the extracted material to correspond with pure natural NPY.

Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas.

Galanin immunoreactivity was measured by RIA, using antibodies directed against both the non-C- and C-terminal positions of porcine galanin, in tissue extracts of normal adrenals and pheochromocytomas and also in the plasma of normal subjects and patients with pheochromocytomas. No C-terminal galanin-like immunoreactivity was detected in plasma or tissue, suggesting differences in the amino acid sequence of human compared with porcine galanin. A non-C-terminally directed antibody was, therefore, used to characterize human galanin immunoreactivity by gel permeation chromatography and reverse phase high pressure liquid chromatography and to localize it by immunocytochemistry. The galanin content of whole adrenal gland was 2.6 +/- 0.9 (+/- SEM) pmol/g (n = 5). In contrast, however, pheochromocytomas had much greater concentrations (21 +/- 2.3 pmol/g; n = 16). Gel chromatography and reverse phase high pressure liquid chromatography revealed 2 molecular forms of galanin immunoreactivity with identical elution positions in both normal adrenals and tumors. The concentration of galanin in plasma from both normal subjects and pheochromocytoma patients was below the detection limit of the assay (less than 10 pmol/liter). Using immunocytochemistry, galanin was localized to scattered cells or clusters of tumor cells in 5 of 11 pheochromocytomas and only a few chromaffin cells and cortical nerve fibers in normal adrenals.

Neuropeptides and a neuronal marker in cutaneous innervation during human foetal development.

There is evidence that foetal body movements first occur at 6 weeks gestation, and that the reflex arc is functional at 8 weeks. This correlates with the detection of the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in spinal cord at 10 weeks gestation. However, the development of cutaneous neuropeptide-containing nerves is not well documented in humans. We have investigated the early appearance and distribution pattern of CGRP, SP, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), as well as those of the general neuronal marker protein gene product 9.5 (PGP) in various areas of foetal skin at different gestational ages. PGP-immunoreactive nerves were first seen in the subepidermal plexus at 6 weeks gestational age. Initially, the immunoreactive nerves are thick, club-shaped and distributed in the superficial dermis. Beaded adult-like fibres become more numerous only at later ages (10-12 weeks), and extend from this plexus to penetrate the epidermis. Histologically, the skin of the hand develops faster than that of other body areas and at 9 weeks, more PGP-immunoreactive nerves were seen in the palm than in the dorsum. Primitive sweat glands were first noted in axillary skin at 17 weeks, accompanied by a few PGP-immunoreactive nerves. Occasional, small CGRP-immunoreactive fibres were first noticed in the dermis at 7 weeks, but it was at 17 weeks that the presence of this neuropeptide was unequivocal in the subepidermal plexus. Sparse VIP-, SP- and NPY-immunoreactive fibres were not found until 16-17 weeks gestation, when they were seen in the dermis and around small blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye.

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

Putative dopamine-containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry.

Immunocytochemistry with antibodies to catecholamine synthesizing enzymes has revealed cells in the retina of chick, mouse, hamster, rat, guinea-pig, piglet and marmoset which contain tyrosine hydroxylase but not dopamine beta-hydroxylase. These findings suggest that the cells in question produce dopamine but that catecholamine synthesis does not proceed further to noradrenaline. Tyrosine hydroxylase-containing amacrine cells, located in the innermost part of the inner nuclear layer, were present in all the species studied. Some species showed atypically located amacrine cells in the inner plexiform or ganglion cell layer. In the rodents, the existence of tyrosine hydroxylase-containing interplexiform cells was suggested by the presence of a few short immunoreactive ascending processes. Three different morphological types of putative dopamine-containing cells were classified according to the level of ramification.

Developmental pattern and distribution of nerve growth factor low-affinity receptor immunoreactivity in human spinal cord and dorsal root ganglia: comparison with synaptophysin, neurofilament and neuropeptide immunoreactivities.

Immunocytochemical expression of the low-affinity nerve growth factor receptor was studied in human fetal and adult tissues using the monoclonal antibody ME20.4. In dorsal root ganglia, a few immunoreactive neurons were first detected in nine-week-old fetuses and many more were found in the following weeks of gestation. However, none was present in adult ganglia. The ME20.4-positive cells were larger than neurons immunostained by substance P, calcitonin gene-related peptide or galanin antibodies. In the spinal cord, fibres immunostained by ME20.4 appeared in a characteristic pattern that differed from the spatial and temporal distributions of synaptophysin- and neurofilament-immunoreactive fibres. Those expressing the low-affinity nerve growth factor receptor were only detected in regions containing collaterals of primary sensory axons: (i) in the dorsal funiculus between seven and 18 weeks of gestation; (ii) in a ventrodorsal bundle reaching the ventral horn from weeks 12-14; (iii) in the medial region of the dorsal horn between weeks 12 and 20; (iv) in the superficial layers and lateral portion of the dorsal horn after the 14th week of gestation and also in adult spinal cord. During the fetal period, ME20.4 immunoreactivity was also found in motoneurons and peripheral nerve fibres in the skin, myotomes and gut. Sheaths of peripheral nerves and the adventitia of blood vessels were stained both in fetal and adult tissues. Thus, the low-affinity nerve growth factor receptor is: (i) strongly expressed in the developing human nervous system; (ii) transiently associated with a subset of large primary sensory neurons and with motoneurons; (iii) transiently and sequentially expressed by various groups of sensory afferents to the spinal cord; (iv) permanently expressed by fibres in the superficial layers of the dorsal horn, Clarke's column, nerve sheaths and the adventitia of blood vessels.