Substance P Fibers Research Articles

OR20-4 Expression of the Neuropeptide Substance P and the NK1 Receptor in Aldosterone-Producing Adenomas

Abstract Background Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism (PA) which is the most frequent form of secondary hypertension (1-2). The pathophysiology of PA is quite complex and still incompletely understood. Substance P (SP) encoded by the TAC1 gene belongs to a family of bioactive peptides named tachykinins. A recently published study indicated that SP, released by subcapsular nerve fibres, stimulates aldosterone production through activation of the neurokinin type 1 receptor (NK1R) in the human adrenal gland (3). The aim of our work was to investigate the presence of SP fibres and the NK1 receptor in a large series of APA in order to assess the potential role of this peptide in the pathophysiology of PA. Methods APA tissues were studied by molecular and histological approaches. Expression of SP and NK1 receptor was searched for in a series of 51 APA by RT-Q-PCR and immunohistochemistry. Results Quantitative RT-PCR data indicated that adenomas strongly express TAC1 mRNA. Immunohistochemistry showed the presence of SP-positive nerve fibres in APAs tissues. SP was also detected in a subpopulation of adenomatous cells. Adenomas strongly express mRNA encoding the NK1 receptor. The distribution of the NK1 receptor within APAs is similar to that of aldosterone synthase (CYP11B2), suggesting that SP may regulate aldosterone secretion by APAs. Conclusion These results suggest that SP and its NK1 receptor may be involved in the pathophysiology of aldosterone hypersecretion by APAs.

Innervation of nociceptors in intact human menisci along the longitudinal axis: semi-quantitative histological evaluation and clinical implications

BackgroundThe mechanism of pain after meniscus injury remains unknown. After injury, some individuals suffered from acute pain, while others suffer from delayed pain. A precise nociceptor distribution pattern may provide the answer to this question.MethodsTwenty-two intact menisci (paired medial and lateral menisci) were obtained from 11 patients with a mean age of 28.45 years. All menisci were sectioned into five parts: the anterior horn, anterior body, middle body, posterior body, and posterior horn. Two paired menisci were stained by a modified gold chloride method. All other specimens were stained by H&E staining and were subjected to immunohistochemical staining to detect substance-P (SP). Under a microscope, measurements were made in 10 consecutive visual areas at 400x magnification. SP-positive fibres were determined using a three-grade scale, and the mean number of SP-positive fibres was assessed.ResultsNerve fibres and nociceptors stained by H&E and modified gold chloride were found mainly in the vascular outer third of the menisci as observed under a microscope; the positive area was wider in the anterior and posterior horns. There were more SP+ fibres in the anterior horn and posterior horn than in the anterior body, middle body, or posterior body (p < 0.05). Regarding the bodies, the mean number of substance-P fibres was greater in the anterior body or posterior body than in the middle area (p < 0.05). No significant differences were found between the number of substance-P nerve fibres in the anterior horn vs the posterior horn or in the anterior body vs the posterior body of all menisci (p > 0.05). No significant differences were observed in the same location between the paired medial and lateral menisci in all areas of the menisci (p > 0.05).ConclusionThe density of nociceptors decreased along the longitudinal axis of the meniscus from both horns to the middle part of the body, which may guide future diagnostic methods and rehabilitation protocols.

Cutaneous neurogenic inflammation in the sensitized acupoints induced by gastric mucosal injury in rats

BackgroundIn acupuncture practice, the most important step is to confirm the location of a sensitized acupoint which reflects a diagnosis and can be stimulated with a specialized needle to treat the disease. Abnormal symptoms such as hyperalgesia or allodynia at the sensitized acupoints in patients with visceral disorders are considered to be in relation with referred pain and neurogenic inflammation. Yet, limited study has investigated the cutaneous neurochemical changes of the sensitized acuponits.MethodsThe resent study developed an animal model of gastric mucosal injury (GMI) by HCl administered into the stomach of the rats. Evans Blue (EB) dye was applied by injection of tail vein after mucosal damage to observe the neurogenic plasma extravasation dots in the skin of the rats. The EB dots extravagated in the skin were compared with locations of acupoints. Immnohistochemistry analysis was used to detect the expression of calcitonin gene-related peptide (CGRP)- or substance P (SP)-labeled nerve fibers, histamine (HA)-, serotonin (5-HT)-, and tryptase-labeled cells in EB dots. Images were recorded and analyzed by Confocal imaging system and Olympus Image Processing Software.ResultsThe results showed that GMI resulted in neurogenic plasma extravasation in the skin of the acupoints over the back and abdomen, which mostly occurred in the T9-11 dermatomere. The EB extravasation dots appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than that in regions beside EB dots and in the control, mostly distributed in the nerve fibers around both the vessels and root of hair follicle. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA around the vessels in areas of the EB dots.ConclusionsOur results indicates that the mechanism of EB extravasation in the skin of the acupoints induced by GMI are closely related to neurogenic inflammation, and that the high expression of local allergic substances and nociceptive neuropeptides in the local skin including SP, CGRP, HA, 5-HT, and mast cell tryptase may be the underlying mechanism of the acupoint sensitization.

Distribution and Origin of VIP-, SP-, and Phospholipase Cβ2 -Immunoreactive Nerves in the Tongue of the Bullfrog, Rana catesbeiana.

Previous studies have found a few intralingual ganglionic cells that were immunoreactive to vasoactive intestinal polypeptide (VIP) in the frog. A recent study reported a large number of such cells, and the possibility of the release of substance P (SP) from these. The aim of the present study was to investigate the distribution, origin, and colocalization of VIP- and SP- immunoreactive nerves in the tongue of the bullfrog, R. catesbeiana. In addition, the study also examined the colocalization of SP and phospholipase Cβ2 (PLCβ2 ) in the tongue and jugular ganglion. VIP immunoreactivity was seen in unipolar cells that were sparse in nerve bundles in the submucosal and muscle layers. The density of VIP-immunoreactive cells was approximately 4.8 cells/mm(3) . Their fibers terminated in the vicinity of the epithelial basal layer of the fungiform papillae. SP immunoreactivity was not seen in the VIP-immunoreactive cells, but was observed in pseudounipolar cells in the jugular ganglion. The SP fibers terminated close to the free surface, showing spindle- and button-like profiles. Transection of glossopharyngeal nerve resulted in the persistence of VIP-immunoreactive cells and the disappearance of SP-immunoreactive fibers in the tongue. SP immunoreactivity was co-expressed with PLCβ2 in both the tongue and jugular ganglia. No PLCβ2 immunoreactivity was seen in cells comprising the epithelial taste disk. These findings indicate that the origin of VIP nerve fibers are unipolar cells in the tongue, and SP and PLCβ2 fibers originate from pseudounipolar cells that may be able to release SP primarily in the jugular ganglion. Anat Rec, 299:929-942, 2016. © 2016 Wiley Periodicals, Inc.

The Distribution of Substance P and Kisspeptin in the Mediobasal Hypothalamus of the Male Rhesus Monkey and a Comparison of Intravenous Administration of These Peptides to Release GnRH as Reflected by LH Secretion

Substance P (SP) was recently reported to be expressed in human kisspeptin/neurokinin B/dynorphin (KNDy) neurons and to enhance KNDy neuron excitability in the mouse hypothalamus. We therefore examined (1) interactions of SP and kisspeptin in the mediobasal hypothalamus of adult male rhesus monkeys using immunofluorescence, and (2) the ability of SP to induce LH release in GnRH-primed, agonadal juvenile male monkeys. SP cell bodies were observed only occasionally in the arcuate nucleus (Arc), but more frequently dorsal to the Arc in the region of the premammillary nucleus. Castration resulted in an increase in the number of SP cell bodies in the Arc but not in the other regions. SP fibers innervated the Arc, where they were found in close apposition with kisspeptin perikarya in the periphery of this nucleus. Beaded SP axons projected to the median eminence, where they terminated in the external layer and intermingled with beaded kisspeptin axons. Colocalization of the two peptides, however, was not observed. Although close apposition between SP fibers and kisspeptin neurons suggest a role for SP in modulating GnRH pulse generator activity, i.v. injections of SP failed to elicit release of GnRH (as reflected by LH) in the juvenile monkey. Although the finding of structural interactions between SP and kisspeptin neurons is consistent with the notion that this tachykinin may be involved in regulating pulsatile GnRH release, the apparent absence of expression of SP in KNDy neurons suggests that this peptide is unlikely to be a fundamental component of the primate GnRH pulse generator.

Role of Neuronal and Non-Neuronal Transient Receptor Potential Vanilloid Type 1 and Sensory Neuropeptides in DSS-Induced Peripheral Inflammatory Changes

BACKGROUND & AIMS: The activation of transient receptor potential vanilloid type 1 (TRPV1) leads to release of sensory neuropeptides such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) from the nerve endings, and these neuropeptides in turn initiate the biochemical cascade known as neurogenic inflammation. TRPV1 channels and the sensory neuropeptides are expressed not only in sensory nerve fibers but also in non-neuronal cells in the mucosa of large intestine. The aim of this study was to investigate alteration of the sensory neuropeptides and TRPV1 channels in mucosa of DSS-induced colitis mice and to characterize their non-neuronal cells. METHODS: Colitis was induced by 3% dextran sulfate sodium (DSS) solution given as drinking water for 7 days in C57BL/6 mice. To evaluate visceral nociception, colorectal distension was performed in mice treated with vehicle or BCTC on day 7 of DSS treatment. Immunohistochemical analysis was performed using rectal tissues of mice. SP, NKA, CGRP, 5-HT, F4/80, keratin, TNF-α, CD11c, and CD4 were detected by indirect staining with their specific antibodies. TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: TRPV1 antagonist BCTC significantly attenuated the visceral hyperalgesia to control level in DSS-induced colitis model. We compared the number of the nerve fibers and non-neuronal cells expressing TRPV1 channels and sensory neuropeptides in normal and DSS-induced colitis model mice. The number of TRPV1and CGRP-expressing nerve fibers is significantly increased in colitis model. The non-neuronal TRPV1 cells were markedly increased but non-neuronal CGRP-expressing cells were not observed in colitis model. No significant alteration in the number of SP and NKA positive nerve fibers was detected. On the other hand, SP and NKA positive cells were drastically increased in colitis model. Next, double labeling studies were carried out to characterize TRPV1-, SP-, and NKA-expressing cells under inflammatory state. We found two types of non-neuronal TRPV1-expressing cells in mucosa of colitis model. One is keratin positive cells located upper part of mucosa. Another is TNF-α and/or F4/80 positive cells located middle part of the mucosa. Both types of TRPV1-expressing cells did not colocalize with CD4 and CD11c. SP and NKA positive cells almost completely colocalized with 5-HT in experimental colitis model mice. CONCLUSION: These results suggest that TRPV1immunopositive epithelial cells and macrophage are associated with visceral hyperalgesia in colitis.

Morphological characteristics and peptidergic innervation in the carotid body of spontaneously hypertensive rats.

We examined morphological characteristics of the carotid body of spontaneously hypertensive rats (SHR), those of age-matched normotensive Wistar rats (NWR), and age-matched genetically comparable Wistar Kyoto rats (WKY). We examined the distribution and abundance of four different regulatory neuropeptides: substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the carotid bodies of these three strains of rats. The carotid bodies of SHR were larger than those of NWR and WKY. The values of the long axis of the carotid bodies of SHR were significantly larger (1.3 times) than those of NWR and WKY. In the carotid bodies of SHR, the percentage of relatively large vessels was similar to that of the carotid bodies of WKY, although the carotid bodies themselves were significantly larger than in WKY. The density of VIP varicose fibers in the carotid bodies of SHR was lower than in the carotid bodies of WKY, although the density of SP, CGRP and NPY fibers was similar to that of the carotid bodies of NWR and WKY. These findings suggested that VIP was unrelated to enlargement of the carotid body of SHR, but it might modify the sensitivity of chemoreceptors in the carotid body.

The expression of SP and CGRP in the temporomandibular joints of the rats undergone emotional stress

Objective To observe the substance P(SP) and calcitonin gene-related peptide(CGRP) expressions in the temporomandibular joints(TM J) of the rats undergone emotional stress and explore the relationship between emotional stress and temporomandibular joint disorder(TMD). Methods Ninety SD rats were averagely randomly divided into emotional stress ( ES ) group( n = 30 ), electric foot-shocked (FS) group ( n = 30 ) and control (CON) group( n = 30). The emotional stress was induced by communication box. The TMJ tissues in ES and CON groups were removed after 1, 3 and 5 weeks of emotional stress for scanning electron microscope ( SEM ) test and immunohistochemistry test. The SP and CGRP expressions were examined with SABC immunohistochemistry and then analyzed by image analysis system. Results The expressions of SP and CGRP had significant difference after 1 ,3 and 5 weeks emotional stress ( SP: 124.5 ± 16.9,185.6 ± 1.8 and 193.5 ± 3.5, respectively; CGRP: 185.9 ±5.3, 112.5 ±5.2 and 174.3 ±5.3 ,respectively) (P＜0. 05 ). The SEM results showed that there was a series of structural change on the condylar surface after emotional stress. Conclusion The SP and CGRP energy nerve fibers take part in the TMJ pathological process undergone emotional stress. Key words: Substance P; Calcitonin gene related peptide; Temporomandibular joint; Emotional stress

Substance P (SP) and vasoactive intestinal polypeptide (VIP) in the lower uterine segment in first and repeated cesarean sections

The authors studied the presence of substance P (SP) and vasoactive intestinal polypeptide (VIP) and their related fibers in the lower uterine segment (LUS) in 133 women undergoing cesarean sections (CS) during active labor. These were divided into 2 groups: women undergoing repeat or first CSs. Specimens were collected from the LUS and were evaluated by light microscopy and by immunohistochemistry, for the morphometrical quantification of the SP and VIP fibers in the LUS. The SP amount was higher in the post-CS scar, while the VIP amount decreased: nerve fibers contained an SP amount of up to 13 ± 2.6 C.U., while nerve fibers contained a VIP amount of up to 7 ± 1.9 C.U. The SP amount counts 10 ± 1.5% of the total Bodian fibers, and the ratio of the VIP is 10 ± 1.8% of their total amount. In normal conditions only 6.61 C.U. of the Bodian surface is occupied by SP related nerve fibers in contrast to 6.63 C.U. of the total surface by VIP; the amount of SP increased up to 13 ± 2.6 C.U., while it decreased in the LUS previous scars. The SP levels are higher in repeat CS, while the VIP levels are reduced in the LUS. The increase of SP is probably linked to the attempt to achieve cervical ripening in a post-CS LUS, with the possible consequences of dystocia during vaginal birth after CS. Nevertheless, the decrease of VIP probably affects the relaxation of the internal uterine orifice, compromising the LUS formation and cervical ripening.

T1668 Mast Cell Proteases Stimulate Neurogenic and Non-Neurogenic Mucosal Chloride Secretion in Guinea Pig Distal Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

T1667 Canonical Transient Receptor Potential Channels (TRPC Channels) in Regulation of Mucosal Chloride Secretion in Guinea Pig Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

Depletion of peptidergic innervation in the gastric mucosa of streptozotocin-induced diabetic rats

Autonomic neuropathy affecting the gastrointestinal system is a major presentation of diabetic neuropathy. Changes in the innervation of gastric mucosa or muscle layers can contribute to gastrointestinal symptoms. The present study investigated this issue by quantitatively analyzing the immunohistochemical patterns of the gastric innervation in rats with streptozotocin (STZ)-induced diabetes. In control rats, calcitonin gene-related peptide (CGRP) and substance P (SP) (+) nerve fibers appeared in the gastric mucosa and muscle layers. Double immunohistochemical staining showed that immunoreactivities for SP and CGRP were co-localized with a pan-neuronal marker protein gene product 9.5. Both SP (+) nerve fibers (p<0.001) and CGRP (+) nerve fibers (p<0.005) were decreased in the gastric mucosa within 4 weeks of diabetes; the reduction persisted throughout 24 weeks. Diabetic rats treated with insulin did not show decrease of SP or CGRP (+) fibers in the mucosa 4 weeks after STZ injection (p>0.05). There was no significant change in SP (+) nerve fibers (p>0.05) or CGRP (+) nerve fibers (p>0.05) of the gastric muscle layers. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of SP and CGRP mRNA in the thoracic dorsal root ganglia were similar between diabetic and control animals (p>0.05). Qualitative and quantitative ultrastructural examinations on the gastric mucosa documented unmyelinated nerve degeneration. These results suggest the existence of gastric sensory neuropathy in STZ-induced diabetes, and this pathology provides a foundation for understanding diabetic gastropathy.

Distribution of a splice variant of choline acetyltransferase in the trigeminal ganglion and brainstem of the rat: Comparison with calcitonin gene‐related peptide and substance P

Rat trigeminal ganglion neurons have been shown to contain a splice variant of choline acetyltransferase (pChAT). Here we report the distribution pattern of pChAT-containing afferents from the trigeminal ganglion to the brainstem, compared with that of calcitonin gene-related peptide (CGRP) and substance P (SP), by use of the immunohistochemical techniques in the rat. Most of CGRP(+) SP(+) ganglion cells contain pChAT, whereas half of the pChAT(+) ganglion cells possess neither CGRP nor SP. In the brainstem, pChAT(+) nerve fibers are found exclusively in the trigeminal and solitary systems, although the distribution pattern differs from that of CGRP(+) or SP(+) fibers. First, the ventral portion of the principal sensory nucleus contains many pChAT(+) fibers, with few CGRP(+) or SP(+) fibers. Because this portion receives projections of nociceptive corneal afferents, a subpopulation of pChAT(+) CGRP(-) SP(-) primary afferents is most probably nonpeptidergic nociceptors innervating the cornea. Second, the superficial laminae of the medullary dorsal horn, the main target of nociceptive afferents, contain dense CGRP(+) and SP(+) fibers but sparse pChAT(+) fibers. Because pChAT occurs in most CGRP(+) SP(+) ganglion cells, such sparseness of pChAT(+) fibers implies poor transportation of pChAT to axon branchlets. Another important finding is that pChAT(+) axons are smooth and nonvaricose, whereas CGRP(+) or SP(+) fibers possess numerous varicosities. Our confocal microscopy suggests colocalization of these three markers in the same single axons in some brainstem regions. The difference in morphological appearance, nonvaricose or varicose, appears to reflect the difference in intraaxonal distribution between pChAT and CGRP or SP.

Increase in sensory neuropeptides surrounding the Achilles tendon in rats with adjuvant arthritis

The Achilles tendon in rats with adjuvant arthritis was analyzed by radioimmunoassay (RIA) and semi-quantitative immunohistochemistry for the occurrence of two sensory neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and a sensory modulating peptide, galanin (GAL). The tissue concentration of SP and CGRP in the Achilles tendon and its envelope, i.e. the paratenon and bony insertion, as assessed by RIA was increased by 22% and 71%, respectively, compared to normal controls, whereas the level of GAL was unchanged. Semi-quantitative immunohistochemistry applied to different regions of the tendon in arthritic rats disclosed an increased occurrence of SP and CGRP positive nerve fibers in the paratenon and bone tendinous junction, whereas GAL fibers were only increased at the bone tendinous junction. Notably, neither neuropeptides nor inflammatory cells were seen in the tendon proper. The increased occurrence of SP and CGRP in the tendon envelope presumably reflects inflammatory actions, whereas that of GAL implies an endogenous anti-inflammatory response. The observed SP and CGRP upregulation in the paratenon and bony insertion suggests a pathophysiological role in paratenonitis and enthesitis often seen in patients with rheumatoid arthritis. Presumably Achillodynia originates in the tendon envelope rather than the tendon proper. The observations could be used to define new pharmacological targets for mitigating symptoms from tendons in rheumatoid arthritis and possibly also in other disorders. Whether a neuronal pathogenic mechanism underlies tendon overuse disorders in non-arthritic tendinopathies and the development of degeneration, i.e. tendinosis, remains to be studied.

Cutaneous and sympathetic denervation in neonatal rats with a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 gene

The mutilated-foot rat ( mf rat) is an autosomal recessive mutant with characteristic digit deformities in adult animals, and this phenotype mimics many aspects of human sensory neuropathy. The genetics of mf rats was recently elucidated. To understand whether the genotype is responsible for cutaneous denervation before clinically overt mutilation in adult mf rats, we investigated skin innervation in postnatal day 7 (P7) mf rats and compared the patterns with P7 wild-type rats. The mf rat carries a G→A mutation in the gene encoding the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 ( Cct4). In the footpad skin of P7 mf rats, there was a >90% loss of epidermal nerves (0.7–7.9% of P7 wild-type rats) as indicated by neuronal markers including protein gene product 9.5 (PGP 9.5), growth-associated protein 43 (GAP43), calcitonin gene-related peptide (CGRP), and substance P (SP). The epidermis of hairy skin in hind feet was completely denervated in mf rats as well. Compared with an approximately 80% reduction in the size of dermal nerve fascicles and a parallel loss of nerve fibers, the nearly complete absence of epidermal innervation suggests further sensory nerve degeneration at the level of nerve terminals in the epidermis. In contrast, the loss of epidermal nerves in the abdominal skin of mf rats was less extensive than that in the footpad skin of mf rats; CGRP (+) and SP (+) fibers were moderately reduced (28.3–56.4% of levels of wild-type rats) with normal amounts of PGP 9.5 (+) and GAP43 (+) nerves. Sympathetic innervation as assessed by tyrosine hydroxylase immunoreactivity was absent from the footpad and abdominal skin of mf rats. In conclusion, there is regional skin denervation with diffuse sympathetic denervation in P7 mf rats. These results suggest that the mutation in Cct4 underlies cutaneous nerve degeneration in mf rats.

Development and distribution of mast cells and neuropeptides in human fetus duodenum.

To study the developmental regularities and heterogeneity of mast cells (MC) in human fetus duodenum and the distribution and developmental regularities of substance P(SP), calcitonin gene-related peptide (CGRP)-immunoreactive (IR) peptidergic nerves in fetus duodenum, as well as the relationship between MC, SP and CGRP- IR peptidergic nerves. Duodena from 21 cases of human fetus and one term infant were stained by hematoxylin-eosin (HE), toluidine blue (TB) and immunohistochemical avidin-biotinylated peroxidase complex (ABC) method. Lobe-shape intestinal villi in duodenum were already developed at the twelfth week. At the 21st wk, muscular mucosa appeared gradually, and four layers were observed in the wall of duodenum. TB staining showed that the granules in the immature MC were pale violet, while the mature MC were strong violet in color by TB staining. Connective tissue MC (CTMC) appeared occasionally in submucosa and muscular layer of duodenum at the 16th wk. While the mucosa MC (MMC) appeared at the 18th wk. At the 22nd wk, both CTMC and MMC were activated, and distributed in the surrounding blood vessels and ganglions. The verge of some MC were unclear, and showed degranular phenomena. At the 14th wk, SP and CGRP-IR nerve fibers and cells appeared in the myenteric and submucous plexuses in small intestine, and the responses were turn strongly. Neurons were light to deep brown, and nerve fibers were present as varicose and liner profiles. On the corresponding site of serial sections, SP and CGRP immunohistochemical reactions were coexisted in one nerve fiber or cell. Some of MC showed SP and CGRP-IR positive staining. There are two heterogeneous kinds of MC in duodenum, MMC and CTMC. MC might play an important role in regulating blood circulation and sensation.

Peptidergic innervation in the rat carotid body after 2, 4, and 8 weeks of hypocapnic hypoxic exposure.

The distribution and abundance of neuropeptide-containing nerve fibers were examined in the carotid bodies of rats exposed to hypocapnic hypoxia (10% O2 in N2) for 2, 4, and 8 weeks. The carotid bodies after 2, 4, and 8 weeks of hypoxic exposure were enlarged by 1.2-1.5 times in the short axis, and 1.3-1.7 times in the long axis in comparison with the normoxic control ones. The enlarged carotid bodies contained a number of expanded blood vessels. Mean density per unit area (10(4) microm2) of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive fibers was transiently high in the carotid bodies after 4 weeks of hypoxic exposure, and decreased significantly to nearly or under 50% after 8 weeks of hypoxic exposure. Density of vasoactive intestinal polypeptide (VIP) immunoreactive fibers increased significantly in all periods of hypoxic exposure observed, and was especially high in the carotid bodies after 4 weeks of hypoxic exposure. Density of neuropeptide Y immunoreactive fibers was unchanged in the carotid bodies during hypoxic exposure. These characteristic changes in the density of SP, CGRP, and VIP fibers in the carotid bodies after 4 weeks of hypoxic exposure suggest that the role of these neuropeptide-containing fibers may be different in the carotid bodies after each of three periods of hypoxic exposure, and that the peptidergic innervation after 8 weeks of hypoxic exposure may show an acclimatizing state.

Mast cells, nerves and neuropeptides in atopic dermatitis and nummular eczema.

The association between mast cells and sensory nerves and the distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) were studied immunohistochemically in lesional and nonlesional skin of 26 atopic dermatitis (AD) and 23 nonatopic nummular eczema (NE) patients. Mast cell-nerve contacts were counted morphometrically and confirmed by confocal laser scanning microscopy. Neuropeptide positivity was assessed semiquantitatively. Dermal contacts between mast cells and nerves were increased in number in both lesional and nonlesional samples of AD and NE when compared to those in normal controls, although only the values in lesional AD reached statistical significance ( P<0.05). Nerve-mast cell contacts in the basement membrane zone were seen practically only in lesional NE. SP and CGRP fibres were prominently increased in lesional samples when compared to their nonlesional controls both in AD and NE in the epidermis and in the papillary dermis. In both AD and NE, only small differences were found regarding VIP positivity in lesional and nonlesional biopsies. The epidermis was devoid of VIP positivity. In conclusion, SP and CGRP but not VIP fibres were more frequent in lesional than in nonlesional papillary dermis of both AD and NE. Since mast cells are also increased in number in lesions of AD and NE, they are able to maintain neurogenic inflammation through activation by SP and CGRP. The increased SP/CGRP nerves in the epidermis of AD and NE lesions may stimulate keratinocytes to release cytokines which affect various cell types enhancing inflammation.

Neuronal plasticity in relation to nociception and healing of rat achilles tendon

Nerve regeneration and the occurrence of three neuropeptides; i.e. substance P (SP), calcitonin gene related peptide (CGRP) and galanin (GAL), were studied during healing of tendon rupture in the rat by semi-quantitative immunohistochemistry. The neuronal findings were related to nociception as assessed by hindpaw withdrawal latencies at thermal and mechanical tests. Experimental rupture of rat Achilles tendon––normally devoid of nerves––elicited extensive nerve ingrowth into the rupture site in the early phase of healing followed by almost complete fiber disappearance (weeks 12–16). The ingrowth of SP and CGRP positive fibers, seen already at weeks 1–2, was associated with increased nociception. Subsequently, the occurrence of GAL positive fibers at weeks 4–6 was associated with decreased nociception. An even stronger relationship to nociception during healing was observed when the rate of change in neuropeptide expression instead of the expression in absolute terms was considered, according to the “cascade” formula of SP ′+CGRP ′−GAL ′. It may prove that the observed temporal occurrence of different neuropeptides reflects a role of the peripheral nervous system in regulating synchronously nociception and healing.

Distribution of myelinated and unmyelinated nerve fibers and their possible role in blood flow control in crotaline snake infrared receptor organs.

We used transmission electron microscopic montages to examine the composition of nerve bundles serving the infrared pit organs of two species of crotaline snakes, Agkistrodon blomhoffii and A. brevicaudus. In the three main bundles, the myelinated fibers totaled 2,200-3,700, and unmyelinated fibers 2,400. We also discovered for the first time two accessory bundles composed almost entirely of unmyelinated fibers running alongside the main bundles, containing an average total of 3,300 unmyelinated fibers vs. an average of 10 myelinated fibers. Thus, the average total of unmyelinated fibers was nearly twice that of myelinated fibers. To study the nature of the unmyelinated fibers, we did double staining immunohistochemistry with antibodies for substance P (SP) and vasoactive intestinal polypeptide (VIP) in combination with and without capsaicin pretreatment. SP and VIP immunoreactive varicose fibers ran straight toward the center of the pit membrane in parallel with arterioles and venules, and also formed a dense network around the periphery of the membrane. There were three types of fibers: fibers containing only SP, fibers containing only VIP, and fibers containing both peptides. SP-only fibers were distributed singly throughout the pit membrane and in small bundles around the periphery. SP+VIP fibers were distributed sparsely in the pit membrane and around its periphery. VIP-only fibers were distributed throughout the pit membrane and were of smaller diameter than SP and SP+VIP fibers. After treatment with capsaicin, most of the three types of varicose fibers disappeared from the central part of the pit membrane, but those around the periphery remained unaffected. The capsaicin-sensitive fibers may be unmyelinated sensory types, and the unaffected ones may be autonomic nerve fibers.