Early increase in CGRP- and VIP-immunoreactive nerves in the skin of streptozotocin-induced diabetic rats.

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.     

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin

Summary Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.     

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase--antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin- positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (_SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 _ 7.25%, 70.52 _ 9.33% and 41.76 _ 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 _ 6.19% and 59.62 _ 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 _ 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow This revised version was published online in November 2006 with corrections to the Cover Date.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Myenteric plexus of obese diabetic mice (an animal model of human type 2 diabetes)

The myenteric plexus of the gastrointestinal tract was investigated in the obese diabetic mouse, an animal model of human type 2 diabetes. Sections were immunostained by the avidin-biotin complex method, using a general nerve marker, protein gene product 9.5 (PGP 9.5), as well as antibodies to several important neurotransmitters. Computerized image analysis was used for quantification. When diabetic mice were compared with controls, no difference was found in the density of PGP 9.5-immunoreactive (IR) nerve fibres in antrum, duodenum or colon. In antrum and duodenum, diabetic mice showed a decreased number of vasoactive intestinal peptide (VIP)-IR neurons in myenteric ganglia as well a decreased relative volume density in myenteric plexus (though not significantly in antrum, p=0.073). No difference was found regarding VIP-IR nerves in colon. The volume density of nitric oxide synthase (NOS)-IR nerve fibres was decreased in antrum and duodenum of diabetic mice, whereas no difference was found in colon. The density of galanin-IR nerve fibres was decreased in duodenum. Whereas neuropeptide Y (NPY)- and vesicular acetylcholine transporter (VAChT)-IR nerve fibres was increased in density in colon of diabetic mice, no difference was found in antrum and duodenum. Regarding substance P, there was no difference between diabetic and control mice in antrum, duodenum or colon. The present study shows that gut innervation is affected in this animal model of human type 2 diabetes. It is possible that the present findings may have some relevance for the gastrointestinal dysfunctions seen in patients with type 2 diabetes.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat

Summary The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vescicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. III. Distribution of neuropeptides in normal and diabetic (host) pancreas.

This study examines whether there is a change in the pattern of distribution of cholecystokinin-octapeptide (CCK-8), calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the pancreas of streptozotocin (STZ)-diabetic (host) rats after subcutaneous pancreatic transplantation. Varicose CCK-8-immunopositive nerve fibres were observed in the wall of blood vessels of both normal and diabetic host pancreata. The density of CCK-8-immunoreactive varicose nerve fibres appeared to have increased in host rat pancreas. CGRP was demonstrated in many nerve fibres located in the wall of blood vessels of both normal and host pancreas. CGRP, however, seemed to be better expressed in the nerves of host pancreas when compared to normal. The pancreata of both normal and diabetic (host) rats contained numerous NPY-immunopositive varicose nerve fibres located in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of the pancreas of STZ-induced diabetic rats with SPTG. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. VIP-positive nerve fibres were still discernible especially in the interacinar regions of the pancreas of host rats. In conclusion, the pattern of distribution and density of NPY, SP and VIP in the pancreas of STZ-induced diabetic rats with SPTG is similar to that observed in normal pancreas, but the expression of CGRP and CCK-8 seemed to have increased as a result of transplantation and or diabetes.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vesicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Ectatic blood vessels in port-wine stains lack innervation: possible role in pathogenesis.

The innervation pattern of port-wine stains was investigated using indirect immunohistochemistry with antibodies to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), calcitonin gene-related peptide (CGRP), and neurofilament (NF). The pathologically dilated vessels in the middle and deep dermis were found to have defective innervation with only single or no nerve fibers in their vicinity, while other structures in the skin showed a normal density of fibers. NSE- and PGP-like immunoreactive (-LI) nerve fibers were observed innervating vessels with a normal morphology and other structures in the skin, such as sweat glands and hair follicles, as free nerve endings and in nerve bundles. The nerve bundles were often seen to pass the ectatic vessels without giving off any branches. CGRP-LI nerve fibers were detected running toward epidermis, whereas no fibers were found around the ectatic vessels. NF-LI fibers were seen innervating normal vessels in dermis, while in relation to the dilated vessels, no or only occasional fibers were observed. The lack of innervation may be of importance for the development of the disease as a result of decreased tonus of the vessels and/or a loss of neuronal trophic factors.     

Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry

The immunocytochemical distribution of substance P (SP), gastrin releasing peptide (GRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and neuropeptide Y (NPY) was studied in the ovary and the Fallopian tube (oviduct) of rats, guinea-pigs, cows, pigs and humans. Generally, the nerve supply was better developed in the oviduct than in the ovary. GRP fibers were most scarce in all tissues. Nerves containing SP were particularly numerous in the oviduct of rat and guinea-pig, supplying the muscular wall and blood vessels. VIP and PHI coexisted in dense plexuses of nerves, not only around blood vessels but also in the follicular wall and the interstitial gland of the ovary, as well as within the smooth muscle layers and subepithelially in the oviduct. The general distribution of NPY was similar, but these immunoreactive nerves were even more numerous. Sequential staining for dopamine-beta-hydroxylase and NPY together with results of chemical sympathectomy with 6-hydroxydopamine suggested that NPY was stored in the noradrenergic sympathetic nerves.     

The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake,Elaphe obsoleta

Summary Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5–30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1–3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.     

Vitiligo-related neuropeptides in nerve fibers of the skin

Skin distribution of substance P (SP)-, somatostatin (SOM)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (LI) in vitiligo patients was studied by an indirect immunofluorescence technique. Immunocytochemical characteristics of the epidermis, dermal-epidermal junction, papillary and reticular dermis and skin appendages were analyzed in lesional and marginal vitiligo areas, as well as in healthy skin. In healthy pigmented skin, SP-, SOM-, CGRP-, and NPY-LI nerve fibers were observed with specific distributional patterns. In uninvolved vitiligo skin, thin SP-containing fibers were evident in dermal papillae, extending into the epidermis, and SP-LI fibers were seen around blood vessels and sweat glands. SOM-LI varicose nerve fibers were associated with Meissner corpuscles in the dermal papillae, while CGRP-LI was demonstrated in the free subepidermal nerve terminals and in sensory nerve fibers around blood vessels, hair follicles and sweat glands. Autonomic NPY-nerve fibers innervated the eccrine sweat glands and blood vessels. The distribution of these neuropeptides in both marginal and lesional areas of vitiliginous skin was the same as in the skin of healthy control subjects, except for an increased immunoreactivity against NPY and, to a lesser extent, against CGRP in the skin depigmentation lesions. The elevated NPY levels in skin affected by vitiligo suggest that this peptide may serve as a neurochemical marker in the pathogenesis of the disease, thus supporting the neuronal theory of vitiligo.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

Summary Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry

Summary The immunocytochemical distribution of substance P (SP), gastrin releasing peptide (GRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and neuropeptide Y (NPY) was studied in the ovary and the Fallopian tube (oviduct) of rats, guinea-pigs, cows, pigs and humans. Generally, the nerve supply was better developed in the oviduct than in the ovary. GRP fibers were most scarce in all tissues. Nerves containing SP were particularly numerous in the oviduct of rat and guinea-pig, supplying the muscular wall and blood vessels. VIP and PHI coexisted in dense plexuses of nerves, not only around blood vessels but also in the follicular wall and the interstitial gland of the ovary, as well as within the smooth muscle layers and subepithelially in the oviduct. The general distribution of NPY was similar, but these immunoreactive nerves were even more numerous. Sequential staining for dopamine--hydroxylase and NPY together with results of chemical sympathectomy with 6-hydroxydopamine suggested that NPY was stored in the noradrenergic sympathetic nerves.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin. Relation to fibers containing substance P-, somatostatin- and vasocactive intestinalpolypeptide-like immunoreactivity

Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin gene-related immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

Changes in fibre populations of the rat hairy skin following selective chemodenervation by capsaicin

Perineural application of capsaicin results in a selective and permanent reduction in the sensitivity to noxious chemical and heat stimuli and elimination of the neurogenic inflammatory response. The present quantitative immunohistochemical study has been undertaken to reveal the populations of cutaneous afferent nerves that are affected by perineural capsaicin treatment. Areas of intact and chemodenervated skin were determined with the aid of the vascular labelling technique. In sections taken from intact skin areas, staining with antibodies against protein gene product 9.5 revealed a rich epidermal innervation. Fibres immunoreactive for growth-associated protein 43 were also abundant; nerve fibres immunoreactive for substance P and calcitonin gene-related peptide were less numerous. Somatostatin- and RT97-immunoreactive fibres were seen only in the subepidermal layer. In sections taken from skin areas supplied by the sciatic nerve treated with capsaicin 3 days previously, the number of epidermal nerve fibres immunoreactive to protein gene product 9.5, growth-associated protein 43, substance P and calcitonin gene-related peptide was reduced by 90%, 95%, 97% and 66%, respectively. These changes persisted for at least 42 days. The findings reveal that the majority of epidermal axons are capsaicin-sensitive and comprise a chemically heterogeneous population. Reductions in cutaneous fibre populations following perineural capsaicin treatment may result from both the degeneration of sensory axons and the depletion of neuron-specific macromolecules. In addition, most cutaneous nociceptive axons may not use the major sensory neuropeptides substance P and calcitonin gene-related peptide as afferent neurotransmitters.