Neuropeptides in the human intervertebral disc

The innervation of the human intervertebral disc was investigated by immunochemical methods. Immunoreactivity to the general nerve marker protein gene product (PGP 9.5) was found in the outer annulus fibrosus of 11 of 12 discs removed during anterior arthrodesis for back pain. PGP 9.5-immunoreactive fibres ran between and across the collagenous lamellae, both in association with blood vessels and distant from them, and extended at least 3 mm into the disc. No innervation was observed in the nucleus pulposus. Fine fibres (< 1 μm in diameter) immunoreactive to calcitonin gene-related peptide and substance P (neuropeptides located in sensory and possibly nociceptive nerves) were identified in eight and four of the annuli fibrosi, respectively. Nerve fibres immunoreactive to vasoactive intestinal peptide and to the c-flanking peptide of neuropeptide Y were found in the majority of specimens of annulus fibrosus that were examined.     

Immunohistochemical demonstration of nerve fibers in the synovial fold of the human cervical facet joint.

The role of the intra-articular synovial fold as a source of facet joint pain is unclear, because the nature of nociceptive innervation in lumbar synovial folds is controversial, and there have been no such studies in cervical synovial folds. The present study aimed to demonstrate the presence of nerve fibers including nociceptive fibers in synovial folds of human cervical facet joints using immunohistochemistry. Synovial folds of cervical facet joints removed from patients undergoing cervical spine laminoplasty were analyzed immunohistochemically using antibodies to protein gene product 9.5, beta III-tubulin, substance P and calcitonin gene-related peptide. Many nerve fibers immunoreactive for protein gene product 9.5 and beta III-tubulin were demonstrated both around blood vessels and as free fibers in the stroma of the synovial fold. Also. immunostaining showed the presence of free nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the stroma. The presence of putative nociceptive fibers in cervical synovial folds supports a possible role for these structures as a source of cervical facet joint pain.     

Morphological basis for back pain: the demonstration of nerve fibers and neuropeptides in the lumbar facet joint capsule but not in ligamentum flavum.

The innervation of lumbar facet capsule and ligamentum flavum was investigated using antisera to a general neuronal marker protein gene product (PGP) 9.5 and to peptide markers of sensory nerves (calcitonin gene-related peptide [CGRP] and substance P) and autonomic nerves (vasoactive intestinal polypeptide [VIP] and C-flanking peptide of neuropeptide Y [CPON]). In the facet capsule (n = 14), PGP 9.5 and CGRP-immunoreactive nerves occurred in 12 and five specimens, respectively, both around blood vessels and as free fibers in the stroma. Free fibers immunoreactive for substance P or VIP were noted in three and five specimens, whereas in nine specimens there were CPON-immunoreactive nerves located perivascularly. There was no immunoreactivity in the ligamentum flavum. This study provides further evidence that the facet capsule but not the ligamentum flavum has substantial innervation by sensory and autonomic nerve fibers and has a structural basis for pain perception.     

Neurogenic factors in the impaired healing of diabetic foot ulcers

BACKGROUND: We hypothesize that the reduced innervation of skin can be observed both in clinically neuropathic and non-neuropathic diabetic foot ulcers and can contribute to low inflammatory cell infiltration. MATERIALS AND METHODS: Twenty patients with type 2 diabetes and active foot ulcers, without clinical evidence of peripheral sensory neuropathy (n = 12) and with sensory neuropathy (n = 8) were involved in this study. Biopsies from ulcer margin were examined immunohistochemically. RESULTS: Studies revealed presence of protein gene product 9.5 (PGP9.5)+ nerve endings only in reticular dermis in 3 of 12 non-neuropathic subjects, however, regenerating GAP-43+ endings were seen in dermis of almost all specimens. Lack of substance P+ nerve endings was characteristic for both groups. The reduced distribution of calcitonin gene-related peptide+ nerves in epidermis and dermis was seen mainly in neuropathic group. In neo-epidermis lack of nerve growth factor expression was observed in both groups, whereas neurotrophin 3 immunostaining was characteristic for neuropathic specimens (P < 0.03). Expression of trkA and trkC receptors did not differ significantly between groups. Low inflammatory cell infiltration and moderate presence of fibroblasts was characteristic for all studied specimens. CONCLUSIONS: The observed reduction of foot skin innervation and neurogenic factors expression can be correlated with low inflammatory cell accumulation and subsequently leads to the observed chronicity of diabetic foot ulcer healing process in both neuropathic and non-neuropathic patients.     

Changes in innervation of long bones after insertion of an implant: Immunocytochemical study in goats with antibodies to calcitonin gene-related peptide and B-50/GAP-43

In this study, we describe the distribution of fibres that contain calcitonin gene-related peptide in normal bones and in bones that are remodeling after insertion of an implant. With routine histology and antibodies to calcitonin gene-related peptide, small neural and free-running fibres staining positively for calcitonin gene-related peptide were found in the periosteum, endosteum, and cortical bone of the tibia in the goat. In many cases, the free-running fibres were associated with blood vessels that entered the bone through Volkmann's canals. The endosteal blood supply was destroyed as a result of insertion of the implant. The necrotic bone was no longer innervated, as shown by the lack of staining for the antibodies. At 6 weeks, a repair phase started with revascularization and remodeling of the necrotic endosteal bone. During this repair phase, there was increased innervation with fibres containing calcitonin gene-related peptide in the remodeling cavities at the interface between living and necrotic bone. These fibres ended blindly, with many large varicosities, and could be demonstrated by immunostaining with monoclonal antibodies to B-50/growth associated protein-43, an antibody to outgrowing neuronal fibres. The correlative occurrence between extensive sprouting of fibres containing calcitonin gene-related peptide and the remodeling of necrotic endosteal bone suggests that sensory fibres with calcitonin gene-related peptide have a regulatory role in the control of angiogenesis or of bone remodeling associated with the insertion of an implant, or with both processes.     

Calcitonin gene-related peptide,substance P,and tyrosine hydroxylase-immunoreactive innervation of rat bone marrows: An immunohistochemical and ultrastructural investigation on possible efferent and afferent mechanisms

The presence of nerve fibers in bone marrow has been noted by various investigators, and recent developments in immunohistochemistry have enabled differential localization of the intramedullary nerve fibers. Much interest has been devoted to the efferent activities of the afferent fibers, which probably act on the target tissues by secreting a variety of neurotransmitters. The present study aimed to further characterize intramedullary substance P, calcitonin gene-related peptide, and tyrosine hydroxylase-immunoreactive nerve fibers of the rat lower limb by comparing those of the knee, ankle, and tarsal joints. The ultrastructural details of intramedullary calcitonin gene-related peptide-immunoreactive axons were also investigated to provide a morphological basis for their possible efferent actions. Intramedullary calcitonin gene-related peptide and substance P-immunoreactive fibers in the proximal tibia and the knee joint were found to be as reported earlier, but the marrow of the distal metaphysis was also noted to be richly innervated, and the tarsal joints displayed dense innervation at the subchondral regions that underlie the joint cartilage. The articular and intramedullary innervations that function for joint protection might participate in characteristic clinical features of joint damage secondary to the neuropathies. Ultrastructurally, the intramedullary calcitonin gene-related peptide-immunoreactive axons were minimally engulfed by the Schwann cell, and naked intramedullary calcitonic gene-related peptide-immunoreactive axons were noted along an extraordinarily long extension, suggesting much efferent activity.     

Plasticity of peptidergic innervation in healing rabbit medial collateral ligament

Background

Denervation substantially impairs healing of the medial collateral ligament (MCL). Because normal ligaments are sparsely innervated, we hypothesized that neuropeptide-containing neurons would sprout or proliferate after ligament transection, followed by later regression with healing, in a manner analogous to blood vessels.

Methods

We transected the right MCL in 9 mature female New Zealand white rabbits and killed 3 rabbits at 2, 6 or 14 weeks. Alternate sets of 12-mm serial sections of healing MCL scars were examined by fluorescent immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and pan-neuronal marker PGP9.5.

Results

Normal MCLs had few peptidergic fibres located in the epiligament in a perivascular pattern. At 2 weeks, PGP9.5-, SP-and CGRP-positive fibres had increased in the epiligament adjacent to the injury. By 6 weeks, there were increases in CGRP-and PGP9.5-positive fibres in epiligament and scar, with similar but less marked increases in SP-positive fibres. At 14 weeks, there was notable regression of immunostained peptidergic nerve fibres in the scar.

Conclusion

This experiment shows evidence for a remarkable plasticity of ligament innervation after injury, supporting the idea that neuronal factors play a fundamental role in wound healing.     

Sensory nerve ingrowth during bone graft incorporation in the rat

We studied nerve ingrowth into a cancellous bone graft in a bone conduction chamber model in the rat. Before implantation of the chamber bilaterally in the proximal tibiae of 8 Sprague-Dawley rats, a defatted cancellous bone graft from separate donor rats was fitted snugly into each chamber. After 6 weeks, the animals were perfused with Zamboni's fixative and the chambers were harvested. Immunohistochemi-cal detection of nerve fibers was performed in cry-ostat sections, using antisera to protein gene product 9.5 (PGP 9.5), neural growth-associated protein GAP-43/B-50, calcitonin gene-related peptide (CGRP), substance P and C- flanking peptide of neuropeptide Y (CPON). Nerve fibers were found in 10 out of 16 samples in the newly formed bone, and also in the fibrous tissue which had penetrated deeper into the graft. The nerve fibers were mainly of sensory origin, as they showed immunoreactivity for CGRP and GAP-43/B-50. We speculate that the nerve fibers may act as transmitters of nociceptive impulses from the graft, and as transport pathways for neuropeptides that are actively involved in angio-genesis and in the recruitment and activity of osteogenic cell populations from the graft recipient.     

Cell relationship in a Wistar rat model of spontaneous prostatitis.

PURPOSE: Prostatitis in men is a painful, noninfectious inflammatory condition. It is similar to interstitial cystitis which is associated with increased bladder mast cell and sensory nerve fiber density as well as suprapubic pain. Certain strains of rats may provide a useful model for studies of the development of spontaneous prostatitis. We evaluated the time course, and involvement of mast cells and sensory nerve fibers in this process using Wistar rats. MATERIALS AND METHODS: The prostates of 4, 6, 8, 10 and 13-week-old male Wistar rats were examined for the degree of inflammation, innervation, mast cell density and nerve mast cell relationship using histochemical and immunocytochemical studies. Bacterial cultures of tissue were performed at 13 weeks. RESULTS: The inflammatory cell index increased progressively with age. Inflammation was moderate and consisted mostly of lymphocytes and macrophages associated with occasional glandular epithelial necrosis and edema. The density of nerve fibers immunoreacting with the neuronal marker protein gene produce 9.5 increased gradually with age and fibers immuno-positive for the sensory neuropeptide calcitonin gene-related peptide more than doubled by 13 weeks compared with by 4 weeks. The density of visible mast cells declined after 4 weeks in a pattern that corresponded with the increased percent of mast cells undergoing degranulation. For the mast cells with calcitonin gene-related peptide immuno-positive nerve fibers within a distance of 40 microm. distance correlated significantly with the degree of degranulation. Bacterial cultures were negative at 13 weeks. CONCLUSIONS: Our results confirm previous reports of spontaneous prostatitis in Wistar rats and indicate that moderate inflammation may occur in 80% of rats at as early as age 13 weeks. While the correlation of the nerve mast cell axis with mast cell degranulation does not prove our hypothesis of mast cell mediated inflammatory mediator release in the development of nonbacterial prostatitis, it suggests that such a relationship is possible.     

Reinnervation after nerve suture in rat groin flaps

Regeneration of sensory and adrenergic nerves in the skin was studied in rats. The aim was to investigate the effect of reanastomosing the cut nerve ends of the nerve trunk leading to the microvascular groin flap. Reinnervation was demonstrated immunohistochemically using calcitonin gene-related peptide (CGRP) as marker for sensory nerves, neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers for adrenergic nerves and Protein Gene Product 9.5 (PGP 9.5) as general neuronal marker. It was demonstrated that reanastomosing of the nerve trunk was favourable for both the sensory and sympathetic reinnervation of microsurgical flaps. © 1998 Wiley-Liss, Inc. MICROSURGERY 18:1–5, 1998.     

Substance P and calcitonin gene-related peptide expression at the extensor carpi radialis brevis muscle origin: implications for the etiology of tennis elbow.

With use of immunohistochemistry and antibodies to substance P and calcitonin gene-related peptide, nerve fibers showing substance P-like and calcitonin gene-related peptide-like immunoreactivity were demonstrated at the origin of the extensor carpi radialis brevis muscle in patients with tennis elbow (n = 6) and in healthy controls (n = 6). The nerve fibers were distributed in association with a subpopulation of small blood vessels and in nerve bundles but were not distributed in the tunica media-adventitia junction of the arterioles. There were no inflammatory-cell infiltrates and few solitary mast cells. The present study gives further evidence to previous suggestions that tennis elbow is not an inflammatory process in the sense of involving inflammatory cells. Frequent mechanical involvement affects sensory innervation, and substance P and calcitonin gene-related peptide may have various important efferent effects, including microvascular leakage and local edema formation; therefore, the observations from this study constitute a morphological substrate for possible effects of substance P and calcitonin gene-related peptide at the origin of the extensor carpi radialis brevis muscle.     

Pharyngeal branch of the vagus nerve carries intraepithelial afferent fibers in the cat pharynx: An elucidation of the origin and central and peripheral distribution of these components

The presence of a sensory component in the pharyngeal branch of the vagus nerve (PhB), including its peripheral distribution and central projection, was studied by denervation and tracer experiments in the cat. The distribution of nerve fibers immunoreactive to protein gene product 9.5, a sensitive neuronal marker; calcitonin gene-related peptide; and substance P in the pharyngeal epithelium was analyzed in both intact animals and animals subjected to partial denervation by means of sectioning two of the three nerve trunks, the glossopharyngeal nerve, the superior laryngeal nerve, and the PhB, while leaving one intact. The results of this study show that the glossopharyngeal nerve and superior laryngeal nerve carry nerve fibers to the pharyngeal epithelium rostral and caudal to the middle level of the epiglottis, respectively, whereas the PhB carries nerve fibers to the mesopharyngeal epithelium. Tracer experiments, by applying wheat germ agglutinin-horseradish peroxidase to the PhB, demonstrated retrogradely labeled primary sensory neurons in the jugular ganglion and transganglionic labeling of terminals in the interstitial subnucleus of the nucleus of the tractus solitarius. These results indicate that the PhB contains a sensory component that originates from the jugular ganglion, innervates the mesopharyngeal epithelium, and projects to the nucleus of the tractus solitarius.     

Nerve regeneration during patellar tendon autograft remodelling after anterior cruciate ligament reconstruction: An experimental and clinical study

The innervation of the rat and human anterior cruciate ligament, patellar tendon, and patellar tendon autograft after reconstruction of the anterior cruciate ligament was investigated by immunohistochemical and histological methods. A rat model of reconstruction with patellar tendon autograft was evaluated during active graft remodelling (2–16 weeks) and compared with normal ligament and tendon. The knees of 10 patients who had undergone reconstruction with patellar tendon autograft were examined 5–37 months postoperatively (remodelling fully completed) with arthroscopy and biopsy. As a control, biopsies from normal ligament and tendon were obtained from four patients. Nerve fibers were identified using antisera for protein gene product 9.5, a general neural marker. Neuronal regeneration was assessed by the expression of growth-associated protein 43/B-50. The sensory type of innervation was characterized by assessing the distribution of nerves containing the sensory neuropeptides calcitonin gene related peptide and substance P. Immunoreactivity for all neural markers was found in both rat and human anterior cruciate ligament and patellar tendon. Two weeks after reconstruction, the rat autograft was acellular and no innervation could be identified. After 4 weeks, the grafts were viable, and immunoreactivity for protein gene product 9.5, growth associated protein 43/B-50, and calcitonin gene-related peptide was found until the 16th week postoperatively. Immunoreactivity for substance P was found in rat autografts at 4 weeks postoperatively only. All biopsies of human patellar tendon autograft showed signs of the remodelling process being fully completed, with revascularization and a sinusoidal collagen pattern with fibroblast repopulation. Neuropeptide immunoreactivity, however, was not found. The presence of immunoreactivity to sensory neuropeptides in the anterior cruciate ligament and patellar tendon may indicate a nociceptive and neuromodulatory function of these structures. The expression of sensory neuropeptides in the rat patellar tendon autograft suggests a possible involvement of sensory innervation during healing of the graft.     

Patients with lower motor spinal cord lesion: a decrease of vasoactive intestinal polypeptide, calcitonin gene-related peptide and substance P, but not neuropeptide Y and somatostatin-immunoreactive nerves in the detrusor muscle of the bladder.

Specimens of the detrusor muscle of the bladder from four patients with lower motor neurone lesion and three patients with carcinoma of the bladder used as "controls", were studied immunohistochemically for vasoactive intestinal polypeptide, neuropeptide Y, calcitonin-gene related peptide, substance P and somatostatin. The greatest density of nerves in the bladder from "control" patients contained neuropeptide Y, followed in a decreasing order by vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P and somatostatin. Neuropeptide Y- and vasoactive intestinal polypeptide-immunoreactive nerves were found throughout the smooth muscle and the base of the mucosa, while calcitonin gene-related peptide-, substance P- and somatostatin-immunoreactive nerves were found predominantly in nerve bundles with a few single fibres at the base of the mucosa. Vasoactive intestinal polypeptide-, neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves were also located around blood vessels. In patients with lower motor neurone lesion, there was a decrease in the density of vasoactive intestinal polypeptide-, calcitonin gene-related peptide- and substance P-immunoreactive nerves, but there was little change in neuropeptide Y- or somatostatin-immunoreactive nerves. Urinary retention, bladder areflexia and deficient sensation may be directly linked to neuropeptide neuropathy in patients with lower motor neurone lesion.     

Silver impregnation and immunohistochemical study of nerves in lumbar facet joint plical tissue

Impingement of plical synovial tissue in a facet joint could cause pain. Plical tissue was removed during surgery for recurrent disc herniation or spinal stenosis. The presence of nerves was studied with silver impregnation, immunofluorescence, and avidin-biotin-peroxidase complex (ABC) immunostaining. Heterologous antisera to protein gene product (PGP) 9.5, substance P, calcitonin gene-related peptide (CGRP), and galanin were used to stain nerves. After silver impregnation, nerve-like structures were observed perivascularly. Such nerves located close to blood vessels were also immunoreactive for PGP 9.5, a more general cytoplasmic neural marker, whereas only few perivascular small varicosities were seen with antisera to substance P and galanin and none with antiserum to CGRP. In addition, PGP-9.5-, substance-P-, and galanin-immunoreactive nerves were occasionally seen very near to fat globules. Very few peptide-immunoreactive nerve varicosities were seen with immunofluorescence, and none of the PGP-9.5-immunoreactive nerves that were observed with ABC immunostaining were immunoreactive for neuropeptides as well. One mechanism for pain production could be mechanical compression of fatty tissue, but it is considered more likely that nerves in this particular tissue are mainly involved in local vasoregulation and that they are not sensory nociceptive nerves.     

Differences in Innervation and Innervated Neurons between Hip and Inguinal Skin

Pain originating from the hip may be referred to the groin and anterior thigh. We investigated sensory dorsal root ganglion neurons innervating the hip and the inguinal skin in rats using retrograde neurotransport and immunohistochemistry. A retrograde neurotracer Fluoro-Gold™ was injected into the left hip or inguinal skin of rats. Seven days later, we harvested bilateral dorsal root ganglions and counted the number of Fluoro-Gold™-labeled neurons positive for calcitonin gene-related peptide, a marker of nerve growth factor-dependent neurons, or isolectin B4, a marker of glial cell line-derived neurotrophic factor-dependent neurons. In the hip group, Fluoro-Gold™-labeled neurons were distributed throughout the left dorsal root ganglions from T13 to L5, primarily at L1, L2, L3, and L4, and the percentage of calcitonin gene-related peptide-positive neurons was higher than that of isolectin B4-binding neurons. In the inguinal skin group, Fluoro-Gold™-labeled neurons were distributed throughout the left dorsal root ganglions from T13 to L3, primarily at L1, L2, and L3, and the percentage of isolectin B4-binding neurons was higher than that of calcitonin gene-related peptide-positive neurons. These data suggest the sensory innervation pattern and characteristics of the sensory nerve of the rat hip are different from those of inguinal skin. Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.     

The reinnervation pattern of wounds and scars may explain their sensory symptoms.

Anaesthesia, pruritis and pain are common in cutaneous scars. The reinnervation pattern of healing wounds and scars might help to explain these symptoms, as sensory neurotransmitters are known to be mediators of inflammation and healing. We quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds as they matured into scars. Mice underwent 1cm(2) full thickness skin excisions. Wounds were harvested between five and 84 days. Sections underwent immunohistochemical staining for protein gene product 9.5 (PGP9.5) a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localisation and quantification of blood vessels. Nerve fibre density was quantified at multiple sites within wounds. There was no difference in the reinnervation/revascularisation pattern between peripheral and central sites. The density of PGP9.5, CGRP, SP and VWF peaked between 14 and 42 days, and levels of PGP9.5, CGRP and VWF all decreased to approximately those found in unwounded skin by 84 days (mature scar). SP levels, however, remained elevated at approximately twice the density found in unwounded skin. Increased densities of SP and CGRP in healing wounds could explain the unpleasant sensory symptoms of healing wounds.     

Early nerve regeneration after Achilles tendon rupture — a prerequisite for healing? A study in the rat

Nerve regeneration during healing of Achilles tendon rupture in the rat was studied by immunohistochemistry including semi-quantitative assessment. Neuronal markers for regenerating and mature fibers, ie., growth associated protein 43 (GAP-43) and protein gene product 9.5 (PGP 9.5), respectively, were analyzed at different time points (1-16 weeks) post-rupture. In the paratenon, both the ruptured and intact contralateral tendon (control) consistently exhibited immunoreactivity to the two neuronal markers. However, in the proper tendinous tissue only the ruptured tendon showed immunoreactivity to GAP-43 and PGP 9.5. This expression was seen already at week 1 post-rupture to reach a peak at week 6 followed by a successive drop till week 16. Also the occurrence of sensory and autonomic fibers according to immunoreactivity for calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY), respectively, was analyzed. CGRP-positivity was abundantly seen from weeks 2-6 in both perivascular and sprouting free nerve endings in the proper tendon tissue undergoing healing. NPY appeared later, at weeks 6-8 post-rupture around blood vessels mainly located in the surrounding loose connective tissue. Apart from a role in vasoaction (CGRP, vasodilatory; NPY, vasoconstrictory). both neuropeptides have been implicated in fibroblast and endothelial cell proliferation required for angiogenesis. The present study shows that early healing of ruptured tendons is characterized by an orchestrated, temporal appearance of nerve fibers expressing peptides with different actions. The observed pattern of neuronal regeneration and neuropeptide expression may prove to be important for normal connective tissue healing.     

Changes in neuroendocrine elements in bronchial mucosa in chronic lung disease in adults.

BACKGROUND--It is not clear whether there is any association between metaplasia of the bronchial epithelium and changes in the distribution of neuroendocrine cells. This study examined, by immunohistological techniques, the distribution of neuroendocrine cells and juxtamucoscal nerve fibres in bronchial biopsies showing metaplastic changes. METHODS--Bronchial biopsies from 12 subjects with epithelial metaplasia associated with bronchiectasis and diffuse pulmonary fibrosis were examined by conventional light microscopy and immunohistological techniques for protein gene product 9.5 (PGP), chromogranin A and B (CAB), serotonin, vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), calcitonin (CT), and gastrin releasing peptide (GRP). RESULTS--Regions of non-metaplastic epithelium contained numerous PGP and serotonin immunoreactive cells. Sub-populations of these cells displayed CAB, CGRP, CT, and GRP immunoreactivity. Metaplastic epithelium contained only a few weakly stained PGP, serotonin, CAB, GRP, CT and CGRP immunoreactive cells in six cases. Metaplastic epithelium was characterised by a high number of CAB-containing cells in six cases and in these biopsies prominent PGP-containing nerve bundles were seen in the subepithelial layer beneath the metaplastic epithelium. CONCLUSIONS--The distribution patterns of neuroendocrine cells and neuronal elements vary between areas of normal and metaplastic epithelium and within areas of metaplastic epithelium. Neuronal hyperplasia was associated with an increase in the number of CAB-containing cells within the metaplastic epithelium.     

Neuronal regeneration in denervated muscle following sensory and muscular neurotization

Background and purpose?Neurotization of denervated muscles has been shown to improve muscle bulk, but the neuronal regeneration response has not been compared previously in different surgical techniques of neurotization. Thus, using a rat model of experimental skeletal muscle denervation, we studied neuronal regeneration following sensory neurotization by two methods: sensory nerve to motor branch of muscle and direct sensory nerve implantation to muscle.

Material and methods?The lateral head of the gas-trocnemius muscle was denervated in 36 rats, of which the first 12 served as denervated controls. In the second group of 12, the sural nerve was anastomozed to the motor branch of the gastrocnemius muscle (sensory-to-motor nerve neurotization) and in the remaining 12 rats the sural nerve was split into 4 fascicles and embedded into 4 quadrants of the muscle (direct sensory nerve-to-muscle neurotization). Immunohistochemistry was used to examine nerve fibers in muscle containing the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and general neuronal marker protein gene product 9.5 (PGP 9.5).

Results?Semiquantitative analysis showed that, compared to the control side, the number of nerve fibers on the experimental side was highest (p < 0.01) for group III (direct sensory nerve-to-muscle neurotization) for all 3 markers. The difference was 71%, 298%, and 254% for PGP 9.5, CGRP, and SP, respectively.

Interpretation?This method may be a good option for inducing neuronal regeneration in denervated muscles, and has therapeutic implications for prevention of atrophy of denervated muscles and as an adjunct for reconstruction of soft tissue defects.