Immunohistochemical evaluation of substance P and calcitonin gene-related peptide in skin and periosteum after extracorporeal shock wave therapy and radial pressure wave therapy in sheep

OBJECTIVE: To evaluate the effect of focused extracorporeal shock wave therapy (ESWT) and radial pressure wave therapy (RPWT) on immunohistochemical staining for substance P and calcitonin gene-related peptide (CGRP) in the skin and periosteum of sheep. ANIMALS: 36 sheep. PROCEDURES: All 4 limbs of 36 sheep were treated with ESWT, RPWT, or a sham treatment. For 14 days after treatment, at least 2 sheep were euthanized daily and tissue was harvested for histologic evaluation of nerves via staining for substance P and CGRP in the skin and periosteum. RESULTS: No effects of ESWT or RPWT were observed on the number of nerves with stain uptake for substance P or CGRP in the skin or periosteum. CONCLUSIONS AND CLINICAL RELEVANCE: Substance P- and CGRP-containing nerve fibers are not disrupted by EWST or RPWT. Further studies are needed to identify the mechanism of analgesia observed in association with these treatment modalities.     

Immunohistochemical demonstration of chromogranin A in endocrine organs of the rat and horse by use of region-specific antibodies

Chromogranin A (CgA) is an acidic glycoprotein that is co-stored with hormones or neurotransmitters in granular components of endocrine cells and neurons, and released together with them in response to adequate stimulation. In addition to acting as a packaging protein, CgA functions as a precursor molecule that yields several bioactive peptides by proteolytic cleavage. The purpose of this study is to elucidate how different the processing of CgA is among endocrine tissues by immunostaining using multiple region-specific antisera, and to evaluate the availability of region-specific antisera. When various endocrine organs of rats were immunostained with four region-specific antisera against rat CgA (CgA 1-28, 94-130, 296-314, and 359-389), all amine/peptide-secreting endocrine tissues except the pineal body were stained positively. The adrenal medulla and gastric endocrine cells were equally intensely immunoreactive to all four antisera, while the other endocrine tissues, represented by pancreatic islets, showed different staining patterns depending on the antiserum. These results suggest that the processing of CgA differs from tissue to tissue. An antiserum against horse CgA 335-365, corresponding to rat CgA 359-389 which shows the highest concentration in the plasma and urine of the rat, again stained all endocrine tissues of the horse except the pineal body. Therefore, the anti-horse CgA 335-365 serum is useful for immunohistochemical survey of horse CgA, and may make possible the establishment of a CgA assay system for the measurement of CgA in the plasma, urine and saliva.     

Ultrastructure of thyroid C cells in sheep treated with vitamin D3.

Ultrastructural observation was performed in C cells of sheep injected intramuscularly with a dose of 2 million IU of vitamin D3 daily for 10 days, 20 days and 30 days, respectively. After treatment with vitamin D3, hyperplasia and hypertrophy of C cells were noticed mainly at the periphery of the thyroid follicles. Most of hypertrophied C cells degranulated conspicuously and contained many prosecretory granules near the well developed Golgi apparatus which were in the actively secreting and packaging phase of their secretory cycle. The other C cells had prominent lamellar arrays of rough-endoplasmic reticulum and aggregations of free ribosomes in the cytoplasm which were interpreted to be in the actively synthesizing phase of their secretory cycle. Atrophic C cells which contained degenerative organelles in the cytoplasm were occasionally observed among the hypertrophied C cells. The present ultrastructural findings clarified that C cells synthesize and secrete calcitonin continuously due to prolonged hypercalcemia induced by long-term administration of excessive doses of vitamin D3 in sheep.     

Immunohistochemical localization of substance P,calcitonin gene-related peptide,galanin and calcium-binding proteins in trigeminal ganglia of goat (Capra hircus)

The objectives of this study was to provide a quantitative analysis of calcium-binding proteins, calbindin (CB), parvalbumin (PA), substance P (SP), calcitonin gene-related peptide (CGRP) and galanin (GAL), in trigeminal ganglia of goats, to establish whether they exhibit coexistence relationships between each other, and to examine possible colocalization with SP, CGRP and GAL, which have been well characterized according to their distributions in an abundance of large and/or small neurones. CB (12.78%), PA (31.91%), SP (24.63%), CGRP (44.44%) and GAL (3.29%) immunoreactive (IR) cells were observed. About 38.37, 8.7 and 0.73% of CGRP-IR neurones in the trigeminal ganglion were also immunoreacted with SP, GAL and CB, respectively. Almost all SP-IR cells are labelled with CGRP (approximately 92.52%), whereas only 16.02 and 0.44% of SP-IR neurones colocalized with GAL and CB. Approximately 4.65 and 1.10% of the CB-IR cells were found to contain CGRP and SP immunoreactivity, respectively. Conversely, no CB-IR cell exhibited GAL immunoreactivity. In addition, all the GAL-IR cells showed CGRP and SP immunoreactivity. The number of CB-, PA-, SP-, CGRP- and GAL-IR neurones in goat trigeminal ganglion are abundant than that of other animals. These results elucidate that the goat differs from other mammalian species in the distribution and localization of neurochemical substances in trigeminal ganglia, and suggest that this difference may be relevant to the morphological characteristics of cerebral vasculatures such as epidural rete mirabile of goat.     

Immunohistochemical localization of chromogranin A in normal tissues from laboratory animals

To analyze the distribution of Chromogranin A in endocrine cells of various species of laboratory animals (dog, gerbil, guinea pig, hamster, monkey, mouse, and fetal, neonatal, and adult rats), normal tissues were stained immunohistochemically with polyclonal anti-bovine Chromogranin A antiserum (SP-1). Selected tissues (pituitary, adrenal, thyroid, parathyroid, pancreas, brain, peripheral nerve, stomach, small and large intestine, bone marrow, spleen, thymus, lymph node, and liver) from these species and from the rabbit were stained with two monoclonal anti-human Chromogranin A antibodies (LK2H10 and PHE5) to compare the immunoreactivities of the monoclonal antibodies and polyclonal antiserum. Staining with the polyclonal antiserum (SP-1) resulted in a broader spectrum of immunoreactivity but had more nonspecific background staining than either monoclonal antibody. Immunoreactivity and staining intensity with SP-1 varied between species, but most endocrine tissues (pituitary cells in the anterior and intermediate lobes, thyroid "C" cells, adrenal medulla, parathyroid, pancreatic islets, and enterochromaffin cells) from most species stained positively. In some species, pancreatic alpha cells stained more intensely, and two populations of adrenal medullary cells with different staining intensities were observed. Sciatic nerve (axonal area) was immunoreactive with monoclonal antibodies and/or the polyclonal antiserum in several species. The spectrum of immunoreactive tissues from fetal and neonatal rats increased with age. There was good cross-reactivity between species with SP-1, but not with either LK2H10 or PHE5. These results indicate that many endocrine cells with secretory granules in laboratory animals express Chromogranin A and that a polyclonal antiserum, such as SP-1, is more sensitive in detecting this protein in various species than monoclonal antibodies such as LK2H10 or PHE5.     

Calbindin D-28k, parvalbumin and calcitonin gene-related peptide immunoreactivity in the canine spinal cord

This study was conducted as a comparative analysis of the immunohistochemical localization of calbindin D-28k, parvalbumin and the calcitonin gene-related peptide (CGRP) in the cervical through the sacral spinal cord of mongrel dogs, to reveal any distinct patterns of distribution and possible involvement in spinal processing. In laminae I and II of the substantia gelatinosa, both calbindin D-28k and CGRP showed strong immunoreactivity, with calbindin D-28k being positive in both cells and fibres, while CGRP was positive in fibres only. Parvalbumin and CGRP immunoreactive cells were widely distributed in various nuclei and lower motor neurones in the ventromedial horn. In addition, the lower motor neurones expressed CGRP as well as parvalbumin, but not calbindin D-28k. These results are generally consistent with previous reports, and the co-localization of parvalbumin and CGRP may explain the functional improvement of lower motor neuron disease.     

降钙素基因相关肽在鸭腔上囊胚胎及胚后发育过程中的表达

应用免疫组织化学技术研究降钙素基因相关肽在鸭腔上囊胚胎及胚后发育过程中的表达特征.结果显示,降钙素基因相关肽强阳性细胞出现于20 d胚龄至胚后14周龄的滤泡间上皮,20 d胚龄至胚后17周龄的小结相关上皮,新生雏至胚后14周龄的滤泡皮质,24 d胚龄至胚后14周龄的滤泡髓质,20 d胚龄至胚后14周龄的肌层和小动脉平滑肌.结果说明,降钙素基因相关肽表达于鸭腔上囊的不同部位及不同发育阶段;在腔上囊退化期,降钙素基因相关肽的表达表现出明显的变化特征,这可能是导致腔上囊退化的原因之一;滤泡间上皮与小结相关上皮在神经肽表达方面存在差异.     

The coexistence of calcitonin gene-related peptide and substance P in pericellular arborization and satellite cell of goat trigeminal and nodose ganglia

Pericellular arborization is reported to be the self-regulating structure in sensory ganglia. Although the calcitonin gene-related peptide (CGRP) or substance P (SP) immunoreactive pericellular arborization appeared in the sensory ganglia, there was no available information that CGRP and SP colocalize in this structure. As the attempts to resolve the question described above, the present study was undertaken to identify the coexistence of CGRP and SP in pericellular arborizations of the goat nodose and trigeminal ganglia by double immunohistochemistry. As the results show, CGRP immunoreactivity was present in every pericellular arborization containing SP immunoreactivity in trigeminal ganglia, however, pericellular network containing CGRP or SP immunoreactivity was not present in nodose ganglia. Unexpectedly, a few small satellite elements were observed to contain intense CGRP and SP immunoreactivity at the periphery of CGRP and SP immunoreactive neurones in nodose ganglia. Therefore, these results suggest that CGRP and SP coexist in pericellular arborizations, and that satellite cell as well as pericellular arborization may be involved in intraganglionic regulation of goat sensory ganglia.     

The distribution of nerve fibres immunoreactive for vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and dopamine beta-hydroxylase in the normal equine larynx

The autonomic innervation of the mammalian respiratory system is complex, and involves a wide variety of peptide and non-peptide neurotransmitters which will have an important role in normal laryngeal function and the response to disease. This innervation has been partially described in the horse airway and lung, but there is no information on the equine larynx. This paper describes the expression and distribution of nerve fibres immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and the adrenergic enzymatic marker dopamine beta-hydroxylase (DBetaH) in the mucosa of the equine larynx. The overall relative density of nerve fibres immunoreactive for the different antigens was VIP>CGRP>SP>DBetaH. There were differences in the distribution of nerve fibre types, although each antigen was found in nerve fibres adjacent to blood vessels and mucous glands. VIP -like immunoreactivity (VIP -Li) was particularly extensive in association with mucous glands. SP - and CGRP -like immunoreactivity (SP -Li, CGRP -Li) were also seen close to the epithelium, with occasional nerve fibres coursing beneath and between the epithelial cells. Fragments of SP -Li and CGRP -Li fibres were also present in large nerve fibre bundles and ganglionic cell clusters, but not in the neurons themselves. The density of nerve fibres immunoreactive for DBetaH was very low and restricted to blood vessels and mucous glands. There was marked variation in the density of nerve fibres at the different sites, with the greatest density, particularly for VIP, over the arytenoid cartilage. Immunoreactive nerve fibres were less plentiful over the epiglottis, and the density of all types of nerve fibres was low over the cricoid cartilage. Overall VIP -Li nerve fibres were the most plentiful.     

Distribution of substance P and calcitonin gene-related peptide in the spinal cord of Cavalier King Charles Spaniels affected by symptomatic syringomyelia

The causes of clinical signs associated with syringomyelia in the Cavalier King Charles Spaniel (CKCS) are incompletely understood. In this study we compared expression of two pain-related neuropeptides: substance P (SP) and calcitonin gene related peptide (CGRP), in the spinal cord dorsal horn of normal dogs with that in CKCS with and without clinical signs of syringomyelia. There was a decrease in expression of both peptides in CKCS with 'symptomatic' syringomyelia that was also associated with significant asymmetry in SP-I and similar, though non-significant, asymmetry in CGRP-I compared with other groups. The asymmetric distribution of these pain-related peptides may be a consequence of syrinx-associated damage to grey matter but may also play a role in generation of pain.     

Expression of vasoactive intestinal peptide, calcitonin gene-related peptide, substance P, and intermediate neurofilaments in nasal mucosal nerve fibers of horses without nasal disease

OBJECTIVE: To determine the distribution of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and intermediate neurofilaments in nasal mucosa of horses. ANIMALS: 6 horses without evidence of nasal disease. PROCEDURE: Full-thickness nasal tissue specimens were obtained from the rostral portion of the nasal septum at necropsy, and fluorescence immunohistochemistry was performed to assess mucosal distribution of nerve fibers. RESULTS: Nerve fibers with CGRP-like immunoreactivity (CGRP-Li) formed a dense subepithelial network, and a large number of fibers were found coursing between epithelial cells. Fibers with CGRP-Li were also associated with blood vessels and mucous glands. Fibers with SP-like immunoreactivity (SP-Li) had a similar distribution and density. In contrast, there were few fibers with VIP-like immunoreactivity. Fibers containing intermediate neurofilaments were prominent and appeared as large nerve fiber bundles mainly adjacent to the nasal septum but also close to mucous glands and within the lamina propria. Intermediate neurofilaments were also identified in single nerve fibers at all sites, but the density of fibers with intermediate neurofilaments did not match that of fibers with CGRP- or SP-Li. CONCLUSIONS: The density and distribution of nerve fibers containing SP- or CGRP-Li in nasal mucosa of horses was similar to that reported for other species. However, expression of VIP in nerve fibers was low. Antibodies against intermediate neurofilaments identified many nerve fibers in nasal mucosa of horses but did not appear to identify small diameter fibers expressing SP or VIP.     

Immunohistochemical demonstration of keratin in canine neuroepithelioma

Morphological features and immunoreactivity for cytokeratin (CK), glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) of three canine neuroepitheliomas and three canine ependymomas were investigated. Neuroepitheliomas were in three German shepherds as intradural-extramedullary solitary masses, with spinal cord displacement between T10 and L2. Histologically, they contained tubules and acini, lined by epithelial cells with focal squamous metaplasia, rosette-like structures, and polygonal to spindle-shaped cells between tubules. Acini were empty or filled with a homogeneous, eosinophilic periodic acid-Schiff (PAS)-positive material. Mitotic indices varied from low to moderate. Ependymomas occurred in the third (two cases) and fourth ventricle in adult boxers. Histologically, they were composed of cells with an ill-defined, scant amphophilic cytoplasm, with a central round euchromatic nucleus; cells formed pseudorosettes, with a central fibro-vascular stroma. Neuroepitheliomas stained for CK, but ependymomas did not. Both failed to stain for GFAP, NSE, or phosphotungstic acid hematoxylin (PTAH). Thus, antibodies to cytokeratin are useful to distinguish neuroepitheliomas from ependymomas.     

Galanin-immunoreactive cells and their relation to calcitonin gene-related peptide-, substance P- and somatostatin-immunoreactive cells in rat lumbar dorsal root ganglia

We report upon the distribution of galanin-immunoreactive (GAL-IR) cells in the lumbar dorsal root ganglia (DRG) of the rat, and upon the distribution of GAL-IR cells, which also contain calcitonin gene-related peptide (CGRP)-, substance P (SP)- and somatostatin (SOM)-immunoreactivity. Neuropeptide-immunoreactive lumbar DRG cells were 55.8% for CGRP, 12.7% for SP, and 6.5% for GAL in lumbar DRG cells. There was no significant difference between the right and left DRGs (L1-L6) for any neuropeptide-immunoreactive cell (P < 0.01). In terms of size distribution, CGRP-immunoreactive cells were identified below 1500 microm2, and SP-, and GAL-IR cells below 600 microm2. Neuropeptide immunoreactive cells showed various immunoreactivities in the cytoplasm according to each neuropeptide. CGRP and SP immunoreactive cells were colocalized with GAL immunoreactive cells in the serial sections about 83.3 and 60% respectively, but SOM colocalizing with GAL-IR cells were not in evidence. The current results confirm and extend previous results, and show that neuropeptides can coexist in single sensory neurones of the rat DRG. In addition, our results demonstrate that the normal distribution of some neurotransmitters modulating sensory action in Wistar Kyoto rat, make this model more prone to develop neuropathic pain than Sprague-Dawley rat.