Angiotensin II type 2 receptors facilitate reinnervation of phenol-lesioned vascular calcitonin gene-related peptide-containing nerves in rat mesenteric arteries

The present study was designed to investigate involvement of angiotensin II (Ang II) type 2 receptors (AT2 receptors) in restoration of perivascular nerve innervation injured by topical phenol treatment. Male Wistar rats underwent in vivo topical application of 10% phenol around the superior mesenteric artery. After phenol treatment, animals were subjected to immunohistochemistry of the third branch of small arteries, Western blot analysis of AT2 receptor protein expression in dorsal root ganglia (DRG) and studies of mesenteric neurogenic vasoresponsiveness. Ang II (750 ng/kg/day), nerve growth factor (NGF; 20 microg/kg/day) and PD123,319 (AT2 receptor antagonist; 10 mg/kg/day) were intraperitoneally administered for 7 days using osmotic mini-pumps immediately after topical phenol treatment. Losartan (AT1 receptor antagonist) was administered in drinking water (0.025%). Phenol treatment markedly reduced densities of both calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and neuropeptide Y (NPY)-LI-containing fibers. NGF restored densities of both nerve fibers to the sham control level. Coadministration of Ang II and losartan significantly increased the density of CGRP-LI-fibers but not NPY-LI-fibers compared with saline control. The increase of the density of CGRP-LI-fibers by coadministration of Ang II and losartan was suppressed by adding PD123,319. Coadministration of Ang II and losartan ameliorated reduction of CGRP nerve-mediated vasodilation of perfused mesenteric arteries caused by phenol treatment. The AT2 receptor protein expression detected in DRG was markedly increased by NGF. These results suggest that selective stimulation of AT2 receptors by Ang II facilitates reinnervation of mesenteric perivascular CGRP-containing nerves injured by topical phenol application in the rat.     

Innervation and functional changes in mesenteric perivascular calcitonin gene-related peptide- and neuropeptide Y-containing nerves following topical phenol treatment

We have previously shown that age-related reduction of innervation and function in mesenteric perivascular calcitonin gene-related peptide-containing vasodilator nerves takes place in spontaneously hypertensive rats. The present study was performed to investigate innervation and functional changes in perivascular calcitonin gene-related peptide- and adrenergic neuropeptide Y-containing nerves after topical treatment with phenol, which damages nerve fibers, around the rat superior mesenteric artery. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) or saline (sham rats) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the 3rd branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 3 through day 14. The innervation levels of calcitonin gene-related peptide-like immunoreactivity containing fibers and neuropeptide Y-like immunoreactivity containing fibers were markedly reduced on day 3 to day 14 and on day 5 to day 14 after the treatment, compared with those in sham-operated rats, respectively. In perfused mesenteric vascular beds isolated from phenol-treated rats, adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide nerve-mediated vasodilation in response to periarterial nerve stimulation (2-12 Hz) were significantly decreased on day 3 and day 7. Neurogenic release of norepinephrine in phenol-treated rats on day 7 was significantly smaller that that in sham-operated rats. Nerve growth factor content in the mesenteric arteries of phenol-treated rats was significantly lower than that in sham-operated rats. Administration of nerve growth factor using osmotic mini-pumps for 7 days after the phenol treatment resulted in greater density of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity fibers than in phenol-treated rats and restored decreased vascular responses to periarterial nerve stimulation. These results suggest that topical phenol-treatment of the mesenteric artery effectively induces functional denervation of perivascular nerves, which can be prevented or reversed by nerve growth factor treatment.     

Release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive vasodilator nerves in the rat mesenteric artery

The effect of perivascular nerve stimulation (PNS) and capsaicin treatment on the release of calcitonin gene-related peptide (CGRP) was examined by radioimmunoassay (RIA) in isolated, perfused rat mesenteric arteries. In the preparation precontracted by methoxamine and treated with guanethidine, an adrenergic neuron blocker, PNS and capsaicin induced vasodilator responses and increase of CGRP-like immunoreactivity (CGRP-LI) in the perfusate in a frequency-dependent manner. The CGRP-LI released by capsaicin was identified as CGRP and its oxidized form by high-performance liquid chromatography coupled with RIA. After the tissue was treated with capsaicin, PNS didn't cause both the vasodilator response and the increase of CGRP-LI in the perfusate. These findings suggest that CGRP plays a neurotransmitter role in capsaicin-sensitive vasodilator nerves in rat mesenteric arteries.     

肾血管性高血压时脑与肠系膜动脉壁CGRP能神经纤维分布的变化

目的：观察肾血管性高血压时中枢与外周动脉壁CGRP（calcitonin gene-related peptide）能神经纤维分布的变化,探讨CGRP与肾血管性高血压的关系.方法：应用免疫组织化学SABC法观察双肾双夹肾血管性高血压大鼠不同时期（术后4、8、12周）脑动脉壁和肠系膜动脉壁CGRP能神经纤维密度的变化.结果：高血压各组CGRP能神经纤维密度均高于对照组,有显著性差异（P＜0.01）;高血压组12周CGRP能神经纤维密度高于高血压组4周,有显著性差异（P＜0.01）;对照组CGRP能神经纤维较纤细,多沿血管长轴走行,高血压组CGRP能神经纤维较粗大,呈网状分布.结论：随着血压的升高,中枢与外周动脉壁CGRP能神经纤维密度增加,这可能是机体产生的一种代偿性保护机制,起着调节血压的作用.     

Inhibitory actions of adenosine differ between ear and mesenteric arteries in the rabbit

In isolated ear and mesenteric arteries of rabbit, adenosine inhibited nerve-mediated contractions to a similar extent. However, the amplitude of the excitatory junction potentials evoked by perivascular nerve stimulation was increased by adenosine in the ear artery and decreased in the mesenteric artery. Outflows of noradrenaline and its metabolite, 3,4-dihydroxyphenylglycol, evoked by perivascular nerve stimulation were increased and decreased by adenosine in the ear and mesenteric arteries, respectively. Adenosine hyperpolarized the smooth muscle cells, by increasing potassium conductance of the membrane, with no relation to the endothelial cells. The hyperpolarizing action of adenosine was stronger in the ear artery than in the mesenteric artery. The inhibition of the nerve-mediated contraction by adenosine may be mainly due to postjunctional events in the ear artery and prejunctional events in the mesenteric artery.     

Inhibitory actions of adenosine differ between ear and mesenteric arteries in the rabbit

In isolated ear and mesenteric arteries of rabbit, adenosine inhibited nerve-mediated contractions to a similar extent. However, the amplitude of the excitatory junction potentials evoked by perivascular nerve stimulation was increased by adenosine in the ear artery and decreased in the mesenteric artery. Outflows of noradrenaline and its metabolite, 3,4-dihydroxyphenylglycol, evoked by perivascular nerve stimulation were increased and decreased by adenosine in the ear and mesenteric arteries, respectively. Adenosine hyperpolarized the smooth muscle cells, by increasing potassium conductance of the membrane, with no relation to the endothelial cells. The hyperpolarizing action of adenosine was stronger in the ear artery than in the mesenteric artery. The inhibition of the nerve-mediated contraction by adenosine may be mainly due to postjunctional events in the ear artery and prejunctional events in the mesenteric artery.     

Ultrastructure of NADPH diaphorase-positive nerve fibers and their terminals in the rat cerebral arterial system

To investigate how perivascular NO synthase (NOS)-containing nerves in the cerebral arterial system are involved in controlling the cerebral circulation, we observed the ultrastructure of NOS-containing nerve fibers and their terminals by means of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. We also observed the correlation between NADPH-d stained perivascular nerves and the perivascular sympathetic nerves, by means of double staining with NADPH-d histochemistry and tyrosine hydroxylase (TH) immunohistochemistry at the light microscopic level. NADPH-d-positive nerve fibers showed dense distribution mainly in the rostral portion of the circle of Willis and proximal portions of its main branches, where some of the NADPH-d-positive fibers coexisted with TH-positive fibers in a single nerve bundle. NADPH-d-positive nerve fibers were unmyelinated and had close contact with NADPH-d-negative myelinated and unmyelinated nerve fibers in a single nerve bundle, and NADPH-d-positive nerve terminals also existed closely with NADPH-d-negative nerve terminals. The number of NADPH-d-positive nerve terminals and their ratio to all other terminals were significantly higher in the rostral portion of the circle of Willis and the proximal portion of its branches, than the caudal portion of the circle of Willis and the distal portion of its branches. Nerve terminals were observed to locate within 250 nm from the basal lamina of arterial smooth muscle cells in the rostral portion of the circle of Willis and proximal portion of its branching arteries. The present observation confirmed that NOS-containing nerve fibers truely innervate the smooth muscle cells of the arterial wall in the circle of Willis and its main branches. Close contact between NADPH-d-positive and -negative nerve fibers and terminals in these arterial portions may indicate that NOS-containing perivascalar nerves may work to modulate the rest of the other perivascular nervous system, such as the sympathetic nerves, to regulate the homeostasis of the arterial tonus.     

Blood vessels in liver metastases from both sarcoma and carcinoma lack perivascular innervation and smooth muscle cells.

Hepatic arterial infusion (HAI) chemotherapy as treatment for human colorectal liver metastases is promising, but not entirely satisfactory. Improved drug delivery during HAI may be achieved by manipulating the different control mechanisms of normal versus tumour blood vessels. The peptidergic/aminergic innervation of vessels in normal liver and in two animal models of liver metastasis (Lister Hooded rat with syngeneic MC28 sarcoma; athymic (nude) rat with human HT29 carcinoma) was investigated to assess the suitability of these models for future pharmacological studies. Normal liver and metastases were studied immunohistochemically for the presence of protein gene product 9.5 (PGP), neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and substance P (SP). Perivascular innervation was also examined by transmission electron microscopy. In Lister rat normal livers, perivascular immunoreactive nerve fibres containing PGP, NPY, TH, CGRP and SP were observed around the interlobular blood vessels near the hilum and in the portal tracts. The highest density was seen for PGP, followed in decreasing order, by NPY, TH, CGRP and SP. VIP-immunoreactive nerves were absent. No immunoreactive nerves were observed in the hepatic lobule. In athymic rat livers, the pattern of innervation was similar, except that SP immunoreactivity was more sparse. No perivascular immunoreactive nerves were observed in either MC28 or HT29 tumours. Electron microscopy confirmed the absence of perivascular nerves. Smooth muscle cells were not observed in tumour blood vessel walls. These results are comparable with previous observations on human liver metastases and suggest that the animal models may be suitable for pharmacological studies on vascular manipulation of HAI chemotherapy.     

Multiple neuropeptides in nerves supplying mammalian lymph nodes: messenger candidates for sensory and autonomic neuroimmunomodulation?

By the use of light microscopic (LM) immunohistochemistry, the presence of peptides and of dopamine beta-hydroxylase (DBH) in nerves supplying mammalian (guinea pig, rat, cat, pig, mouse, human) lymph nodes were examined. In all species, lymph nodes of various somatic and visceral regions were found to contain nerve fibers which stained for neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), substance P (SP), calcitonin gene-related peptide (CGRP) or DBH. SP- and CGRP-immunoreactive (ir) fibers completely overlapped and exhibited the widest distribution. They were present in perivascular, paravascular and many non-vascular fibers travelling in close contact with lymphoid cells. In contrast, NPY-ir fibers coincided with those staining for DBH, prevailed in perivascular plexus and only rarely branched off into lymphoid parenchyma. Alternate staining of adjacent sections revealed that SP/CGRP-ir fibers were different from NPY/DBH-ir fibers. The distribution of VIP-ir fibers was identical to that of PHI-ir fibers and partially overlapped with that of ir-NPY/DBH or ir-SP/CGRP fibers. We conclude that the NPY innervation of lymph nodes is sympathetic noradrenergic while nerves coding for co-existing SP and CGRP are most likely of sensory origin. The nerves containing co-existing VIP and PHI may be of heterogenous origin (sensory, cholinergic sympathetic, and/or parasympathetic). We suggest that these distinct sensory and autonomic peptidergic pathways linking the nervous system with the lymph nodes may play a differential role in bidirectional neuroimmunomodulation.     

Paracrine control of mesenteric perivascular axo-axonal interaction

Immunohistochemical study of rat mesenteric arteries showed dense innervation of adrenergic nerves, calcitonin gene-related peptide (CGRP)-containing nerves (CGRPergic nerves), nitric oxide-containing nerves (nitrergic nerves). Double-immunostaining revealed that most CGRPergic or nitrergic nerves were in close contact with adrenergic nerves. CGRPergic and transient receptor potential vanilloid-1 (TRPV1)-immunopositive nerves appeared in the same neurone. In rat perfused mesenteric vascular beds without endothelium and with active tone, perfusion of nicotine, or bolus injection of capsaicin and acetylcholine and periarterial nerve stimulation (PNS) lowered pH levels of out flowed perfusate concomitant with vasodilation. Cold-storage denervation of preparations abolished pH lowering induced by nicotine and PNS. Guanethidine inhibited PNS- and nicotine-, but not acetylcholine- and capsaicin-, induced pH lowering. Pharmacological analysis showed that protons were released not only from adrenergic nerves but also from CGRPergic nerves. A study using a fluorescent pH indicator demonstrated that nicotine, acetylcholine and capsaicin applied outside small mesenteric artery lowered perivascular pH levels, which were not observed in Ca(2+) free medium. Exogenously injected hydrochloric acid in denuded preparations induced pH lowering and vasodilation, which was inhibited by denervation, TRPV1 antagonists and capsaicin without affecting pH lowering. These results suggest that excitement of adrenergic nerves releases protons to activate TRPV1 in CGRPergic nerves and thereby induce vasodilation. It is also suggested that CGRPergic nerves release protons with exocytosis to facilitate neurotransmission via a positive feedback mechanism.     

TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study

Interscapular brown adipose tissue (IBAT), a site of nonshivering thermogenesis in mammals, is neurally controlled. The co-existence of sympathetic and peptidergic innervation has been demonstrated in different brown adipose depots. We studied the morphological profile of IBAT innervation and tested by immunohistochemical methods whether cold and warm stimulation are accompanied by modifications in the density of parenchymal noradrenergic nerve fibers. We also studied the immunoreactivity of afferent fibers—which contain calcitonin gene-related peptide (CGRP) and substance P (SP)<197>in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4°, 20°, and 28°C). Tissue activity was evaluated by studying the immunolocalization of uncoupling protein (UCP-1), a specific marker of brown adipose tissue. Noradrenergic and peptidergic innervation were seen to arise from morphologically different nerves. Fibers staining for tyrosine hydroxylase (TH) were thin, unmyelinated hilar nerves, and CGRP- and SP-positive fibers were in thick nerves containing both myelinated and unmyelinated fibers. Under cold stimulation, noradrenergic neurons produce greater amounts of TH, and their axons branch, resulting in increased parenchymal nerve fibers density. Neuropeptide Y (NPY) probably co-localizes with TH in noradrenergic neurons, but only in the perivascular nerve fiber network. The parenchymal distribution of NPY to interlobular arterioles and capillaries suggests that this peptide must have other functions besides that of innervating arteriovenous anastomoses, as hypothesized by other researchers. The different distribution of CGRP and SP suggests the existence of different sensory neuronal populations. The detection of CGRP at the parenchymal level is in line with the hypothesis of a trophic action of this peptide.     

大鼠脑血管含神经肽Y、含降钙素基因相关肽、含P物质神经的年龄变化

本文应用免疫组织化学（ＡＢＣ）方法，观察了含神经肽Ｙ（ＮＰＹ）、含降钙素基因相关肽（ＣＧＲＰ）、含Ｐ物质（ＳＰ）神经在大鼠完整脑底动脉环分布的年龄变化，结果表明在２周、２月、８月龄大鼠脑底动脉上含ＮＰＹ神经的密度依次降低；含ＣＧＲＰ和含ＳＰ神经的密度依次升高．至１８月龄，含ＮＰＹ神经的密度和８月龄时的接近，而含ＳＰ和含ＣＧＲＰ神经的密度比８月龄时明显降低．大鼠脑血管含肽神经支配的这种增龄变化表现出有利于脑血管扩张的趋势，它可能是脑血管硬化导致脑血流量下降的一种代偿机制。     

Electron-immunocytochemical studies of perivascular nerves of mesenteric and renal arteries of golden hamsters during and after arousal from hibernation

Electron immunocytochemistry was used to examine perivascular nerves of hamster mesenteric and renal arteries during hibernation and 2 h after arousal from hibernation. Vessels from cold-exposed but nonhibernating, and normothermic control hamsters were also examined. During hibernation the percentage of axon profiles in mesenteric and renal arteries that were immunopositive for markers of sympathetic nerves, tyrosine hydroxylase (TH) and neuropeptide Y (NPY), were increased 2–3 fold compared with normothermic and cold control animals. This increase was reduced markedly only 2 h after arousal from hibernation. The small percentage of nitric oxide synthase-1-positive axon profiles found in mesenteric (but not renal) arteries was also increased during hibernation and returned towards control values after arousal. In contrast, the percentage of perivascular axons immunostaining for vasoactive intestinal polypeptide (VIP), a marker for parasympathetic nerves, was reduced in mesenteric arteries during hibernation. There was no labelling of perivascular nerves for substance P in either mesenteric or renal arteries. It is suggested that the increase in percentage of TH- and NPY-immunostained perivascular nerves may account for the increased vasoconstriction associated with high vascular resistance that is known to occur during hibernation. The reduction in the percentage of axons positive for VIP in hibernating animals would contribute to this mechanism since this neuropeptide is a vasodilator.     

Innervation pattern of polycystic ovaries in the women

The aim of the present study was to determine the changes in both the distribution pattern and density of nerve fibers containing dopamine β-hydroxylase (DβH), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SOM), galanin (GAL) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the human polycystic ovaries. In the polycystic ovaries, when compared to the immunoreactions pattern observed in the control gonads, following changes were revealed: (1) an increase in the number of DβH-, VAChT-, VIP- or GAL-immunoreactive (IR) nerve fibers within the stroma as well as in the number of DβH-IR fibers near primordial follicles and medullar veins and venules; (2) a reduction in the number of nerve fibers containing nNOS, CGRP, SOM, PACAP within the stroma and in the numbers of CGRP-IR fibers around arteries; (3) an appearance of SP- and GAL-IR fibers around medullar and cortical arteries, arterioles, veins and venules, with except of GAL-IR fibers supplying medullar veins; and (4) the lack of nNOS-IR nerve fibers near primordial follicles and VIP-IR nerves around medullar arteries and arterioles. In conclusion, our results suggest that the changes in the innervation pattern of the polycystic ovaries in human may play an important role in the pathogenesis and/or course of this disorder.     

The reinnervation and revascularisation pattern of scarless murine fetal wounds

Fetal wounds can heal without scarring. There is evidence that the sensory nervous system plays a role in mediating inflammation and healing, and that the reinnervation pattern of adult wounds differs from that of unwounded skin. Ectoderm is required for development of the cutaneous nerve plexus in early gestation. It was hypothesised that scarless fetal wounds might completely regenerate their neural and vascular architecture. Wounds were made on mouse fetuses at embryonic day 16.5 of a 19.5-day gestation, which healed without visible scars. Immunohistochemical analysis of wound sites was performed to assess reinnervation, using antibodies to the pan neuronal marker PGP9.5 as well as to the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Staining for the endothelial marker von Willebrand factor (VWF) allowed comparison of reinnervation and revascularisation. Wounds were harvested at timepoints from day 1 after wounding to postnatal day 6. Quantification of wound reinnervation and revascularisation was performed for timepoints up to 6 days post-wounding. Hypervascularisation of the wounds occurred within 24 h, and blood vessel density within the wounds remained significantly elevated until postnatal day 2 (4 days post- wounding), after which VWF immunoreactivity was similar between wound and control groups. Wound nerve density returned to a level similar to that of unwounded skin within 48 h of wounding, and PGP9.5 immunoreactive nerve fibre density remained similar to control skin thereafter. CGRP and SP immunoreactivity followed a similar pattern to that of PGP9.5, although wound levels did not return to those of control skin until postnatal day 1. Scarless fetal wounds appeared to regenerate their nerve and blood vessel microanatomy perfectly after a period of hypervascularisation.     

Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat

Calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal polypeptide (VIP) have been proposed to be neurotransmitters/neuromodulators in cerebral perivascular nerve fibers. Here, we present pharmacological and biochemical evidence showing that these peptides have different modes of relaxing cerebral blood vessels in the cat. CGRP causes pronounced relaxation, this occurs simultaneously with stimulation of cyclic adenosine monophosphate (cAMP) accumulation. The strong VIP-induced dilatation is parallelled by cAMP accumulation, albeit of a lower magnitude than with CGRP. The SP-induced relaxation was much weaker than that of CGRP and VIP, and it was not associated with cAMP accumulation. Only at concentrations of SP where maximum relaxation had occurred, was a nonsignificant cAMP accumulation seen. The responses to SP and acetylcholine were absent in arteries where the endothelium had been removed, whereas the relaxations induced by CGRP and VIP persisted.     

Influence of methanandamide and CGRP on potassium currents in smooth muscle cells of small mesenteric arteries

Cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their mechanism of action, however, is complex. Apart from acting on vascular smooth muscle or endothelial cannabinoid receptors, several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene-related peptide (CGRP). In the present study, the direct influence of the cannabinoid methanandamide and the neuropeptide CGRP on the membrane potassium ion (K⁺) currents of rat mesenteric myocytes was explored. Methanandamide (10 μM) decreased outward K⁺ currents, an effect similar to that observed in smooth muscle cells from the rat aorta. Conversely, CGRP (10 nM) significantly increased whole-cell K⁺ currents and this effect was abolished by preexposure to tetraethylammonium chloride (1 mM) or iberiotoxin (100 nM), inhibitors of large-conductance calcium-dependent K (BK_Ca) channels but not by glibenclamide (10 μM), an inhibitor of ATP-dependent K channels. In the presence of the CGRP receptor antagonist CGRP_8-37 (100 nM), the adenylyl cyclase inhibitor SQ22536 (100 μM), or the protein kinase A inhibitor Rp-cAMPS (10 μM), CGRP had no effect. These findings show that methanandamide does not increase membrane K⁺ currents in smooth muscle cells of small mesenteric arteries, supporting an indirect mechanism for the reported hyperpolarizing influence in this vessel. Moreover, CGRP acts directly on these smooth muscle cells by increasing BK_Ca channel activity in a CGRP receptor and cyclic adenosine monophosphate-dependent way. Collectively, these data indicate that methanandamide relaxes and hyperpolarizes intact mesenteric vessels by releasing CGRP from perivascular nerves.     

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.     

Calcitonin gene-related peptide-immunoreactive nerve fibers in mandibular periosteum of rat: evidence for primary afferent origin

Peptidergic neurons may play a role in the local regulation of bone mineralization. The neuropeptide vasoactive intestinal peptide (VIP) increases bone resorption in vitro, while calcitonin gene-related peptide (CGRP) has been shown to inhibit bone resorption in vitro. We have previously reported that sympathetic nerves with VIP-immunoreactivity innervate bone and periosteum. In the present study we sought to determine if CGRP fibers, like VIP fibers, exist in periosteum and what their origin might be. In whole-mount preparations of mandibular periosteum from rat, CGRP- and VIP-immunoreactive (IR) nerve fibers were present as networks within the periosteum. In preparations using two-color immunofluorescence, most CGRP-IR fibers were also immunoreactive for substance P (SP). In rats in which the subperiosteal space subjacent to the mandibular molars was injected with Fast blue or Fluoro-gold, retrogradely labeled cells were seen in ipsilateral trigeminal ganglia, superior cervical ganglia, and nodose ganglia. Individual cells labeled with both CGRP immunoreactivity and retrograde tracer were seen only in the mandibular portion of the trigeminal ganglion. These data suggest that CGRP-IR nerve fibers in periosteum may be of primary afferent origin. Given the reported effects of CGRP on bone mineralization, the present results suggest that primary afferent nerves containing CGRP and SP, as well as sympathetic nerves containing VIP, may play a role in focal bone remodeling.     

Immunohistochemical demonstration of peptidergic nerve fibers associated with the central lacteal lymphatics in the duodenal villi of dogs.

Immunohistochemical demonstration was made of the peptidergic nerves distributed in the central lacteal lymphatics of the canine duodenal villi. The central lacteal-associating nerve fibers were predominantly immunoreactive for both substance P (SP) and calcitonin gene-related peptide (CGRP). Observation of doubly immunostained sections evidenced that both peptides were located in one and the same nerve fibers. The SP/CGRP-immunoreactive fibers were concentrated in the intermediate portion of the villus height. Ultrastructurally, the SP/CGRP-immunoreactive nerve fibers ran closely beneath the endothelial cells of the lacteal, some of them penetrating into the cytoplasm with knob-like swellings. The immunoreactive products were localized to large-cored vesicles in the sub- and intra-endothelial nerves. The occurrence of SP and CGRP in the nerve fibers distributed in the central lacteals which lack smooth muscles implies that these nerves may be sensory in nature. A mechanoreceptive function of the nerves is proposed on the basis of their peculiar knob-like projection into the lacteal endothelium.