Occurrence of a dense plexus of sensory nerve fibers immunoreactive to calcitonin-gene-related peptide in the cauda epididymidis of rats

The occurrence, distribution and ontogeny of nerves displaying calcitonin-gene-related peptide (CGRP)-like immunoreactivity were studied in the male reproductive tract of rats. A marked regional difference in number of CGRP-immunoreactive nerve fibers was observed in the epididymis. The immunoreactive nerve fibers were particularly numerous in the cauda epididymidis, where the nerves were found in the capsular and interstitial connective tissue and further in the smooth muscle layer and the subepithelial connective tissue surrounding the duct. In the remaining portions of the reproductive tract proximal and distal to the epididymis, CGRP-immunoreactive nerve fibers were scarcely found in the connective tissues surrounding the duct, although a small number of the CGRP-immunoreactive nerve fibers was constantly found adjacent to small blood vessels throughout the male reproductive tract. CGRP-immunoreactive nerve fibers in the epididymis were first detected at embryonic day 18 when thin bundles or single fibers were evenly distributed in the interstitial connective tissue of the entire epididymal duct. A marked regional difference in number of CGRP-immunoreactive nerve fibers seen in the adult epididymis was established by postnatal day 14. In the epididymis of young rats treated neonatally with capsaicin, CGRP-immunoreactive nerve fibers were almost completely absent. This finding together with the distribution pattern of CGRP-immunoreactive nerve fibers different from that of the autonomic nerves so far reported strongly suggests that the immunoreactive nerves were sensory in nature.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

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.     

Nerve fiber production by intraocular adrenal medullary grafts: Stimulation by nerve growth factor or sympathetic denervation of the host iris

Summary This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.     

Nerve growth factor-induced stimulation of dorsal root ganglion/spinal cord co-grafts in oculo: enhanced survival and growth of CGRP-immunoreactive sensory neurons

Intraocular co-grafts of rat fetal spinal cord and dorsal root ganglia were used to examine the enhanced survival, growth, and differentiation of sensory neurons by nerve growth factor. E14 lumbar spinal segments were implanted into the anterior eye chamber of capsaicin-pretreated rats. Two weeks later, an E14 dorsal root ganglion was implanted beside the spinal cord graft. Nerve growth factor or vehicle was injected weekly for 4 weeks into the anterior eye chamber. Co-grafts were examined weekly and, at 6 weeks, processed for calcitonin gene-related peptide (CGRP) immunofluorescence. No differences in overall size were determined for the grafts. Co-grafts treated with nerve growth factor contained many more CGRP neurons (19.4 cells/20 microm) that were significantly larger (mean 764 microm2) than neurons from control co-grafts (8.6 cells/20 microm; mean 373 microm2). In co-grafts treated with nerve growth factor, CGRP-immunoreactive fibers were extensive in the dorsal root ganglion, adjacent iris, and spinal cord compared to control co-grafts. A few CGRP-positive motoneurons were observed in the spinal cord, but no differences in number or size of motoneurons were found. The current report demonstrates that spinal cord and dorsal root ganglia can be co-grafted in oculo for long periods of time. Many dorsal root ganglion neurons survive and send peripheral processes into the iris and central processes into the spinal cord under the influence of exogenous nerve growth factor. The intraocular graft paradigm can be of use to further examine the role of neurotrophic factors in regulating or modulating dorsal root ganglion and spinal cord neurons.     

Locus coeruleus fibre growth in oculo induced by trigeminotomy

Locus coeruleus from fetal donors was homologously grafted to the anterior eye chambers of adult rats whose eyes were sympathetically denervated. After intraocular maturation, outgrowth of noradrenaline-containing fibres from the locus coeruleus neurons on the host iris was studied by Falck--Hillarp fluorescence histochemistry.In control animals locus coeruleus grafts produce a halo of noradrenaline-containing nerve fibres around the graft, covering approximately one third of the surface of the host iris. Sensory denervation of host eyes carrying maturated locus coeruleus grafts was produced by intracranial lesions of the trigeminal nerve. Such lesions induced a rapid growth response in the grafted locus coeruleus neurons, leading within three weeks to complete innervation of the host iris. It was concluded that removal of non-sympathetic, non-parasympathetic nerve fibres on the host iris elicits a strong fibre-growth response in the grafted locus coeruleus.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crossing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerves following traumatic injuries and surgical procedures.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

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-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.     

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Summary Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crousing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerved following tramatic injuries and surgical procedures.     

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat

Summary Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland.The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland.The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

A study of skin deformation by optical coherence microscopy

The deformation of rat skin in response to cooling and stimulation of nerve fibers innervating the piloerector and vasoconstrictor muscles has been studied by optical coherence microscopy. It has been found that the deformation of skin upon cooling is due to the contraction of collagen fiber bundles rather than the smooth muscle of vessels or piloerectors.     

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat

Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland. The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland. The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.     

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.