GAP-43 immunoreactivity is widespread in the autonomic neurons and sensory neurons of the rat.

GAP-43 is a membrane-bound phosphoprotein generally associated with axon growth during development and regeneration. Using immunohistochemical and immunoblotting techniques this study shows that GAP-43 is expressed extensively in the unperturbed adult autonomic nervous system. Strong immunoreactivity was seen in the developing and mature enteric subdivision of the autonomic nervous system and in nerves of the iris and various blood vessels. The presence of GAP-43 immunoreactivity in varicose nerve fibres, and a comparison of the labelling pattern of GAP-43 with the nerve associated marker PGP 9.5 suggests that GAP-43 is present in most or all autonomic nerve fibres in these organs. Immunoblotting of gut samples on 10% polyacrylamide gels revealed a single band of approximately 45,000 mol. wt that co-migrated with pure central nervous system GAP-43. Surgical sympathectomy experiments resulting in almost complete elimination of sympathetic fibres did not markedly affect the pattern of GAP-43 immunoreactivity in the iris, indicating that GAP-43 is expressed not only in sympathetic nerves but also in parasympathetic and sensory fibres. These findings show that GAP-43 is expressed extensively in autonomic nerves of the adult rat, at levels comparable to those seen during development. High levels of GAP-43 are not therefore restricted to development and regeneration in this part of the nervous system.     

Involvement of brain-derived neurotrophic factor (BDNF) in the development of periodontal Ruffini endings

The periodontal Ruffini ending has been reported to show immunoreactivity for tyrosine kinase B (trkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the periodontal ligament of the rat incisor. Furthermore, adult heterozygous BDNF-mutant mice showed malformation and reduction of the periodontal Ruffini endings. To investigate further roles of BDNF in these structures, the development, distribution, and terminal morphology of Ruffini endings were examined in the incisor periodontal ligament of heterozygous and homozygous BDNF mutant mice, as well as in the wild-type littermate by immunohistochemistry for protein gene product (PGP) 9.5, a general neuronal marker. A similar distribution and terminal formation of PGP 9.5-immunoreactive nerve fibers was recognized in the periodontal ligament of all phenotypes at postnatal week (PW) 1. At this stage, the nerve fibers had a beaded appearance, but did not form the periodontal Ruffini endings. At PW2, the heterozygous and wild-type mice started to show ramified nerve fibers resembling the mature shape of periodontal Ruffini endings. At PW3, the Ruffini endings occurred in the periodontal ligament of the wild-type and heterozygous mice. While the Ruffini endings of the wild-type mice appeared either ruffled or smooth, as reported previously, most of these structures showed a smooth outline in the heterozygous mice. The homozygous mice lacked the typical Ruffini endings at PW3. In the quantitative analysis, homozygous mice had the smallest percentages of PGP 9.5-immunoreactive areas at the same postnatal periods, but there were no significant differences between wild-type and heterozygous mice during PW1-3. These findings suggest a possible involvement of BDNF during the postnatal development and, in particular, the maturation of periodontal Ruffini endings. Furthermore, other neurotrophins may play a role in the development and/or early maturation of the periodontal nerve fibers, as indicated by the presence of nerve fibers in the BDNF-homozygous mice.     

Distribution of nerve fibers during the development of palatine glands in rats

Background

Salivary gland maturation and function are modulated by the nervous system. Nevertheless, little is known about salivary gland innervation during development, particularly minor salivary glands. This study investigated the development of the innervation of the palatine glands of rat.

Study of innervation, sensory neuropeptides, and serotonin in murine contact allergic skin

Density of nerve fibers, axonal growth, calcitonin gene-related peptide (CGRP), and substance P, and serotonin immunoreactivity as well as concentration were all determined in a murine model of contact allergy. Female Balb/c mice were sensitized on the back with oxazolone and 6 days later challenged with the same antigen on the dorsal surface of the ears, while control mice received the vehicle only. Then, 24 hr postchallenge, one ear was processed for immunohistochemical staining, while the other was frozen and processed for gas chromatography-mass spectrometry or radioimmunoassay (RIA). Protein gene product 9.5 (PGP 9.5) positive nerve fibers showed a tendency to increase in inflamed ears versus control ears in epidermis as well as the dermis. Growth-associated protein-43 (GAP-43) positive fibers in the epidermis were increased (p < .01) in inflamed ears, compared with control ears, as was the case for the dermal fibers, indicating increased axonal growth. Total (epidermis and dermis) numbers of CGRP and substance P positive nerve fibers tended to increase in the inflamed skin in contrast to control skin. In contrast, RIA demonstrated a lower (p < .05) concentration of CGRP in the inflamed ears compared with controls and a tendency for substance P to decrease in concentration in eczematous ears versus controls. There was no difference in serotonin concentration, or in the number of serotonin positive mast cells, between the inflamed and control skin, whereas semiquantification of serotonin positive platelets showed an increase in the inflamed (+/+) compared with control ears (+). Our results indicate that 24 hr after being challenged with the antigen, at the peak of murine skin inflammation, axonal growth, sensory neuropeptides, as well as serotonin may be involved.     

Age-related changes and the possible adaptability of rat jaw muscle spindles: immunohistochemical and fine structural studies

Afferent signals from jaw muscle spindles contribute to the feedback mechanism that regulates mastication. The integrity and adaptability of this proprioceptor to age-related changes of the surrounding structures are therefore essential to maintain an appropriate masticatory function throughout life. In this study, we examined muscle spindles obtained from temporal and masseter muscles of 10-week-, 12-, 18-, and 24-month-old Wistar rats, employing immunohistochemistry for protein gene product 9.5 (PGP 9.5) or growth-associated protein (GAP-43) in addition to transmission electron microscopy, in order to investigate their morphological changes in relation to the effect of aging on the adaptive potential of the receptors. Immunohistochemistry for PGP 9.5 showed virtually similar reactions at sensory nerve terminals in all age groups. On the other hand, immunoreactivity for GAP-43 in the sensory nerve ending of the muscle spindles was found 2 and 3 weeks after birth but became almost undetectable by 10 weeks. However GAP-43 immunoreactions occasionally reappeared in those of spindles in 12- and 18-month old animals, and vanished again by 24 months of age. Electron microscopic observations also revealed age-related morphological changes in the intrafusal muscle fibers of the rats in 12-month and older groups. The extent of degenerative and/or atrophic alterations of intrafusal fibers increased with age and involved the nerve elements of spindles by 24 months. These findings indicate that the adaptation potential of rat jaw muscle spindles is well preserved until middle age, but diminishes in elderly animals. Structural changes of muscle spindles in elderly animals probably contribute to the deterioration of the muscular function.     

Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.     

Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.     

Delayed expression of calbindin D28k during regeneration of the periodontal Ruffini endings of the rat incisor following injury to the inferior alveolar nerve

Expression of calbindin D28k (CB)-like immunoreactivity (-LI) was compared with that of protein gene product 9.5 (PGP 9.5), a general neuronal marker, in the periodontal ligament of the rat lower incisor following resection of the inferior alveolar nerve (IAN). In normal animals, the periodontal nerve fibers showing PGP 9.5-LI formed either Ruffini endings with expanded arborization or thin free nerve endings in the alveolar half of the ligament. Thick CB-like immunoreactive (-IR) nerve fibers terminated in a dendritic fashion in the same region, but thin CB-IR nerve fibers were rarely detected. During the 3 days following resection of the IAN, most of the PGP 9.5-IR and all CB-IR nerve fibers disappeared. Regenerated PGP 9.5-IR nerve fibers appeared around 7 days after resection, in contrast to the very small number of regenerated CB-IR nerve fibers. Around 21-28 days following resection, the number and terminal morphology of regenerated PGP 9.5-IR nerve fibers were comparable to those observed in normal animals, but the number of regenerated CB-IR nerve fibers was still smaller. The terminal morphologies of these regenerated CB-IR nerve fibers showed less expansion compared with normal animals at these post-injured periods. The number of regenerated CB-IR nerve fibers increased gradually to return to normal by 56 days following injury. The delayed expression of CB in the regenerated periodontal Ruffini endings suggests that the functional recovery of periodontal Ruffini endings occurred after the regeneration of periodontal Ruffini endings had been completed.     

Upregulation of the Axonal Growth and the Expression of Substance P and its NK1 Receptor in Human Allergic Contact Dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.     

The distribution of PGP9. 5, BDNF and NGF in the vallate papilla of adult and developing mice

The development and innervation of vallate papillae and taste buds in mice were studied using antibodies against the neuronal marker, protein gene product 9.5 (PGP 9.5), and against nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). PGP 9.5 immunohistochemical studies revealed that the earliest sign of median vallate papilla formation was an epithelial bulge at embryonic day 13 (E13), and at E14, a dense nerve plexus was found within the connective tissue core of the papilla. Thin nerve fibers penetrated the apical and medial trench wall epithelium of the papilla at E16 and a few of these began to invade the lateral trench wall epithelium at E17. At postnatal day 1 (P1), the newly formed taste buds were recognizable and a small number of PGP 9.5-immunoreactive (IR) cells appeared on the medial trench wall epithelium. The number of PGP 9.5-IR taste bud cells then increased gradually and reached the adult level at postnatal week 2. PGP 9.5 immunoreactivity increased systematically with age. NGF and BDNF immunoreactivity was first seen at the boundary between the columnar cells in the apical epithelium of the developing vallate papilla at E13, then in the medial and lateral trench walls at E15 (BDNF) or E18 (NGF). At P1, BDNF immunoreactivity was exclusively present in the newly formed taste buds of the medial trench wall. The number of BDNF-IR taste bud cells then increased gradually, reaching the adult level at P7. Similar degrees of NGF and BDNF immunoreactivity were seen in the developing vallate papilla. In the present study, we found that the vallate papilla was formed prior to its innervation, and we propose that initiation of papilla formation does not require any direct influence from the specific gustatory nerve. We also suggest that neurotrophins in the early developing vallate papillae might act as local tropic factors for the embryonic growth of nerve fibers to induce differentiation of the taste buds.     

Effects of different types of injury to the inferior alveolar nerve on the behavior of Schwann cells during the regeneration of periodontal nerve fibers of rat incisor

The present study reports on different regeneration patterns of axons and Schwann cells in the periodontal ligament of the rat incisor using immunohistochemistry of protein gene product 9.5 (PGP 9.5) and S-100 protein. Three kinds of injury (transection, crush and segmental resection) were applied to the inferior alveolar nerve. In normal animals, PGP 9.5- and S-100-immunoreactivities were detected in the axons and Schwann cell elements of periodontal Ruffini endings, respectively. They were restricted to the alveolus-related part, occurring only rarely in the tooth-related part and in the shear zone (the border between the alveolus-related and tooth-related parts). Both transection and segmental resection caused the complete disappearance of PGP 9.5-immunoreactive nerve fibers in the periodontal ligament, while a small number of them could be found following the crush injury. Regenerating PGP 9.5-reactive nerve fibers appeared at 5 days and 21 days following the transection and segmental resection, respectively. The regeneration of periodontal nerve fibers completed in a period of 21-28 days and 14-21 days following the transection and crush, respectively, but was not completed even at 56 days following the segmental resection. The behavior of Schwann cells during regeneration was similar after the different nerve injuries; spindle-shaped S-100-immunoreactive cells, presumably Schwann cells, appeared in the shear zone and the tooth-related part. These cells disappeared 5-7 days prior to the completion of the regeneration of axonal elements of the periodontal ligament following the transection and crush. Following the segmental resection, in contrast, spindle-shaped S-100-positive cells disappeared from the tooth-related part at 42 days, although the axonal regeneration of periodontal Ruffini endings proceeded even until 56 days. We thus conclude that the duration of the migration of Schwann cells depends on the state of the regeneration of axons.     

Expression of protein gene product 9.5 in epithelioid and conventional malignant peripheral nerve sheath tumors

CONTEXT: Due to the frequent lack of S100 protein expression in malignant peripheral nerve sheath tumors (MPNSTs), especially the epithelioid variant, these tumors are difficult to diagnose without the aid of electron microscopy or a clinical history of neurofibromatosis. METHODS: Protein gene product 9.5 (PGP9.5), a broad neural marker, is expressed in nerve fibers and neurons of both the peripheral and central nervous systems. We compared its expression to that of S100 protein in 16 cases of MPNST. As controls, 6 monophasic synovial sarcomas, 9 leiomyosarcomas, and 5 dermatofibrosarcoma protuberans were included. RESULTS: Expression of PGP9.5 was seen in 15 MPNSTs, with 3 to 4+ positivity in the majority of the cases. Ten cases, 2 epithelioid and 8 conventional MPNSTs, were reactive with PGP9.5, but were negative for S100 protein. Five cases were immunoreactive for both S100 protein and PGP9.5. One case was negative for PGP9.5 but demonstrated focal S100 protein positivity. Expression of PGP9.5 was seen in 4 of 6 synovial sarcomas, 3 of 9 leiomyosarcomas, and none of 5 dermatofibrosarcoma protuberans. CONCLUSION: Although PGP9.5 is not a specific marker for MPNST, it is a more sensitive marker than S100 protein (94% vs 38%). When there is a lack of S100 protein expression and a broad panel of immunostains, such as cytokeratin, epithelial membrane antigen, and smooth muscle actin, yields only focal or equivocal staining, PGP9.5 is a useful diagnostic adjunct in confirming the neural origin of a spindle cell sarcoma.     

Expression of neuropeptides and growth-associated protein 43 (GAP-43) in cutaneous and mucosal nerve structures of the adult rat lower lip after mental nerve section.

The reinnervation of the adult rat lower lip has been investigated after unilateral section of the mental nerve. Rats were sacrificed at 4, 7, 9, 14, 30, and 90 days after the operation. A further group of animals with section of the mental nerve and block of the alveolar nerve regeneration, was sacrificed at 14 days. Specimens were processed for immunocytochemistry with antibodies against PGP 9.5, GAP-43 or neuropeptides (CGRP, SP and VIP). Four days after nerve section, axonal degeneration seems evident in the mental nerve branches and inside skin and mucosa. GAP-43 immunoreactivity is intense in the mental nerve 7 days after nerve section and it reaches its maximal expression and distribution in peripheral nerve fibres at 14 days. At 30 days, the decline in its expression is associated with the increase of PGP9.5-, SP-, and CGRP immunopositivity. VIP is observed only in perivascular fibres at all times observed. Present results suggest that, after sensory denervation of the rat lip, nerve fibres in skin and mucosa remain at lower density than normal. The different time courses in the expression of neuropeptides and GAP-43 suggest a possible early involvement of GAP-43 in peripheral nerve regeneration.     

Immunohistochemical distribution of growth-associated protein 43 (GAP-43) in developing rat nasoincisor papilla

We employed immunohistochemistry of growth-associated protein 43 (GAP-43) to trace the early development of gustatory nerves and alpha-gustducin to demonstrate mature taste buds in the rat nasoincisor papilla (NP). The sequential changes of gustatory structures revealed eight characteristic stages. One, at embryonic day 16 (E16), GAP-43-immunoreactive (IR) nerve fibers were observed in close relation with presumptive taste buds in the lateral apical epithelium on each side of NP; meanwhile, no immunoreactivity could be observed in the papillary epithelium. Two, at E17, fine GAP-43-IR nerve fibers first invaded the apical epithelium of the papilla. Three, at E19, GAP-43-IR nerve fibers were extensive in apical epithelium and colonized in immature taste buds. Four, at E20, GAP-43-IR nerve fibers were first observed in ductal epithelium (lining the medial wall of nasoincisor ducts). Five, at postnatal day 1 (P1), immunoreactive nerve fibers first coincided with immature taste buds in the ductal epithelium. Six, at P3, alpha-gustducin-IR cells identical for mature taste buds were simultaneously demonstrated in both apical and ductal epithelium. Seven, at P14, progressive taste bud proliferation and maturation as well as neural invasion were demonstrated in all regions of the epithelium. Eight, during advanced stage in adult animals, extensive innervation was traced especially in close relation with taste buds. The sequential topographic patterns of NP gustatory structures seem very specific as compared to those in other locations of mammalian gustatory system. The present study reveals that gustatory nerves preceded the development of taste buds. However, further investigations are required to examine such a characteristic model for the neurogenic theory of taste induction.     

Alteration in the expression of heat shock protein (Hsp) 25-immunoreactivity in the dental pulp of rat molars following tooth replantation.

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immunocytochemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes--presumably newly differentiated odontoblasts--at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulp-dentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.     

Localisation and quantitation of autonomic innervation in the porcine heart I: conduction system

This study was prompted by the prospect of transgenic pigs providing donor hearts for transplantation in human recipients. Autonomic innervation is important for the control of cardiac dynamics, especially in the conduction system. Our objective was to assess the relative distribution of autonomic nerves in the pig heart, focusing initially on the conduction system but addressing also the myocardium, endocardium and epicardium (see Crick et al. 1999). Quantitative immunohistochemical and histochemical techniques were adopted. All regions of the conduction system possessed a significantly higher relative density of the total neural population immunoreactive for the general neuronal marker protein gene product 9.5 (PGP 9.5) than did the adjacent myocardium. A similar density of PGP 9.5-immunoreactive innervation was observed between the sinus node, the transitional region of the atrioventricular node, and the penetrating atrioventricular bundle. A differential pattern of PGP 9.5-immunoreactive innervation was present within the atrioventricular node and between the components of the ventricular conduction tissues, the latter being formed by an intricate network of Purkinje fibres. Numerous ganglion cell bodies were present in the peripheral regions of the sinus node, in the tissues of the atrioventricular groove, and even in the interstices of the compact atrioventricular node. Acetylcholinesterase (AChE)-containing nerves were the dominant subpopulation observed, representing 60–70% of the total pattern of innervation in the nodal tissues and penetrating atrioventricular bundle. Tyrosine hydroxylase (TH)-immunoreactive nerves were the next most abundant neural subpopulation, representing 37% of the total pattern of innervation in the compact atrioventricular node compared with 25% in the transitional nodal region. A minor population of ganglion cell bodies within the atrioventricular nodal region displayed TH immunoreactivity. The dominant peptidergic nerve supply possessed immunoreactivity for neuropeptide Y (NPY), which displayed a similar pattern of distribution to that of TH-immunoreactive nerve fibres. Calcitonin gene-related peptide (CGRP)-immunoreactive nerves represented 8–9% of the total innervation of the nodal tissues and penetrating atrioventricular bundle, increasing to 14–19% in the bundle branches. Somatostatin-immunoreactive nerve fibres were relatively sparse (4–13% of total innervation) and were most abundant in the nodes, especially the compact atrioventricular node. The total pattern of innervation of the porcine conduction system was relatively homogeneous. A substantial proportion of nerve fibres innervating the nodal tissues could be traced to intracardiac ganglia indicative of an extensive intrinsic supply. The innervation of the atrioventricular node and ventricular conduction tissues was similar to that observed in the bovine heart, but markedly different to that of the human heart. It is important that we are aware of these findings in view of the future use of transgenic pig hearts in human xenotransplantation.     

糖尿病大鼠背根神经节JNK的表达水平对足部皮肤组织学变化的影响

目的:观察糖尿病(DM)大鼠皮肤组织学改变,探讨背根神经节(DRG)内c-Jun氨基端激酶(JNK)蛋白在其中的可能作用机制。方法:雄性SD大鼠腹腔注射链脲佐菌素制备DM模型,分别取正常对照(control)组、DM 2周(DM2)组、DM 4周(DM4)组和DM 8周(DM8)组大鼠足底皮肤标本进行PGP 9.5免疫组化染色和HE染色,观察皮肤PGP 9.5免疫阳性神经末梢和皮肤组织结构变化;同时采用Western blotting法分别检测上述各组大鼠足底皮肤组织内PGP 9.5蛋白和脊髓腰5~6(L5,6)、骶1(S1)节段DRG内JNK和p-JNK蛋白的表达。结果:大鼠足底皮肤PGP 9.5免疫阳性神经末梢主要分布于表皮基底层和真皮乳头层,与正常对照组比较,DM4组大鼠表皮阳性神经末梢密度减少,分布稀疏,DM8组大鼠表皮阳性神经末梢分布明显减少,神经直径变细,长度变短,走形扭曲;组织学观察可见,DM4组大鼠表皮组织变薄,表皮细胞层次减少,细胞分布及排列不均匀,DM8组大鼠表皮细胞层次明显减少,细胞肿胀模糊,细胞间隙增大,部分表皮缺乏复层排列,真皮层胶原纤维萎缩、变性,皮下脂肪明显减少。蛋白印迹检测发现皮肤组织内PGP 9.5蛋白的表达随病程呈渐进性减弱;同时L5,6-DRG和S1-DRG内p-JNK蛋白的水平呈现渐进性增强趋势。DM大鼠皮肤PGP 9.5免疫阳性神经末梢吸光度值与L5,6-DRG和S1-DRG内的p-JNK蛋白灰度值之间呈负相关(P0.01);与表皮厚度值之间呈显著正相关。结论:DM大鼠皮肤组织和皮肤神经均存在明显形态结构改变,同时存在L5,6-DRG和S1-DRG内p-JNK蛋白的渐进性高表达,DM大鼠皮肤PGP 9.5免疫阳性神经末梢密度变化与L5,6-DRG和S1-DRG内p-JNK蛋白水平的变化之间呈负相关;与表皮厚度的变化呈显著正相关。皮肤神经形态结构的改变可能与背根神经节细胞JNK/SAPK通路活化有关,阻断或抑制JNK/SAPK通路可以起到延缓糖尿病周围神经病变,减轻皮肤组织病变发生的作用。     

Immunohistochemical demonstration of nerve terminals in the whole hard palate of rats by use of an antiserum against protein gene product 9.5 (PGP 9.5)

Sensory innervation of the entire hard palate was investigated in the rat using serial sections immunostained for protein gene product 9.5 (PGP 9.5), a neuronal marker. PGP 9.5-immunoreactive nerve endings were widely distributed in the hard palate, but the innervation pattern and density differed among portions. They were numerous at papillary protrusions including the incisal papilla, antemolar/intermolar rugae, and postrugal filiform papillae. Immunoreactive free nerve endings gathered at the summits of the connective tissue papillae, some of them entering deeply into the epithelium. Electron microscopy demonstrated that nerves in the postrugal filiform papillae reached the stratum corneum. The atrial region, possibly the most sensitive in the hard palate, showed unique innervation: its anterior part, adjacent to incisors, developed intraepithelial networks of fine and beaded nerves, whereas its posterior part revealed cone-shaped nerve terminals formed on the connective tissue papillae of the atrial folds which comprised two lines of longitudinal flaps. Taste bud-like corpuscles gathered in the medial walls of the incisal canals and in the "Geschmacksstreifen" (taste stripes) present at the most anterior part of the soft palate. The hard palate of the rat is thus richly innervated, and is characterized by region-specific nerve endings which may be involved in mechano- and chemoreception in the oral cavity.     

The three-dimensional distribution of nerves along the entire intrapulmonary airway tree of the adult rat and the anatomical relationship between nerves and neuroepithelial bodies

Using airway microdissection and three-dimensional confocal microscopy techniques in combination with the immunomarkers protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP), we defined the distribution of small afferent nerves fibers and all nerves throughout the intrapulmonary airways, along with the distribution of airway neuroendocrine cells and neuroepithelial bodies. We found (i) the presence of CGRP-and PGP 9.5-positive structures along the entire intrapulmonary airway tree of adult rats, (ii) decreasing nerve density from more proximal to more distal generations of conducting airways, (iii) the presence of nerve fibers in terminal bronchioles, (iv) the asymmetrical distribution of nerves within a single generation of intrapulmonary airway with regard to associated vessels, (v) the frequent interchange of single nerve fibers across epithelial and sub-epithelial compartments without termination, and (vi) a definably intimate relationship between afferent nerves and neuroepithelial bodies (NEBs) (i.e., 58% of NEBs studied were observed to have nerve fibers coursing through them, indicating direct connections). We conclude that the distribution of nervous elements (nerve fibers and neuroendocrine cells) within the intrapulmonary airways is highly heterogeneous, varying between airway levels and locally within a specific airway level.