C.-J. Dalsgaard

Accumulation of T lymphocytes and expression of interleukin-2 receptors in nonrheumatic stenotic aortic valves

OBJECTIVESnCell-specific antibodies were used to identify immunocompetent cells in a comparison of valves from patients who had symptomatic tricuspid aortic stenosis with subjects who had no evidence of valvular heart disease.nnnBACKGROUNDnNonrheumatic valvular aortic stenosis is the most common valvular heart disease among adults. The biologic processes involved in the development of this disease are poorly understood.nnnMETHODSnTricuspid stenotic aortic valves were obtained from 19 patients undergoing surgery for nonrheumatic valvular aortic stenosis, and 10 control valves were collected at autopsy. The valves were fixed in formaldehyde, cryosectioned and stained with antibodies against fibroblasts, endothelial cells, macrophages, T lymphocytes and interleukin-2 receptors. A subset of valves were also analyzed with antibodies against T-helper cells and cytotoxic T cells.nnnRESULTSnStenotic valves were characterized by a basal accumulation of calcium deposits and a cell-rich subendothelial thickening. The immunohistologic analysis indicated that the cells in the subendothelial connective tissue were fibroblasts. T lymphocytes appeared to be the most common cell type in the vicinity of the calcium deposits and were also found close to the endothelial lining of the valves. T-helper cells were more frequent than cytotoxic T cells. Expression of interleukin-2 receptors occurred at the same location as T lymphocytes. Control valves lacked subendothelial thickening and contained only few cells reacting with antibodies against lymphocytes and macrophages.nnnCONCLUSIONSnThe presence of activated T lymphocytes in tricuspid stenotic valves suggests that immunologic mechanisms may be involved in the etiology of nonrheumatic aortic stenosis.

Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A

Antisera were raised in rabbits against the tachykinins neurokinin A (NKA) and substance P (SP). All NKA-antisera tested cross-reacted markedly with NKB, kassinin and eledoisin in radioimmunoassay (RIA), but virtually not with SP and physalaemin. Also when used for immunohistochemistry, one of the NKA-antisera was found to be virtually without cross-reactivity with SP. The most specific SP-antiserum did not cross-react with NKA but to some extent with NKB at the immunohistochemical level. Using these two antisera, the same distribution pattern of immunoreactivity was seen in both the rat substantia nigra and dorsal spinal cord. In neutral extracts of the substantia nigra, all NKA-antisera used for RIA detected a major component which eluted at the position of NKA in reverse phase high performance liquid chromatography, while no or only little immunoreactivity was detected at the position of NKB. A major component of substance P-like immunoreactivity (SPLI) co-eluting with SP and one or two minor SPLI-components were also detected in these extracts. An SP-antiserum, which cross-reacted markedly with physalaemin, detected an additional rather prominent component. In neutral water extracts of dorsal spinal cord the component detected with the NKA-antisera at the position of NKB, as well as one of the SPLI-components not eluting in the position of SP, were much more prominent than in the corresponding extracts of substantia nigra. In acetic acid extracts of both tissues, only one major SPLI-component co-eluting with SP could be detected, while only very small amounts of immunoreactivity eluting at the position of NKA and NKB (dorsal spinal cord only) could be detected using the NKA-antisera. The present results illustrate the importance of the extraction method used in immunochemical studies and demonstrate that the relative proportions of various tachykinins are markedly different in the rat substantia nigra and dorsal spinal cord.

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase-activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat

The distribution of several peptides in cutaneous, muscular and visceral primary sensory neurons was investigated in the adult rat. The fluorescent dye Fast blue was applied to the proximal ends of transected saphenous (cutaneous), gastrocnemius (muscular) and greater splanchnic (visceral) nerves. Sections from corresponding dorsal root ganglia were incubated for simultaneous indirect immunocytochemical demonstration of calcitonin gene-related peptide (CGRP)-, substance P (SP)- or somatostatin (SOM)-like immunoreactivity (-LI) and Fast blue. In addition, the presence of fluoride resistant acid phosphatase (FRAP)-enzyme activity (-EA) in retrogradely Fast blue-labeled saphenous and gastrocnemius nerves was investigated by subsequent enzyme cytochemical analysis. The results revealed the presence of CGRP-LI, SP-LI, SOM-LI and FRAP-EA in cell bodies of primary sensory neurons which project to the saphenous and gastrocnemius nerves. CGRP-LI and SP-LI, but not SOM-LI, were found in splanchnic sensory neurons. The vast majority of the visceral sensory neurons were found to contain CGRP-LI.

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

SummaryUsing 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 Meissners 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.

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

SummaryRat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallell decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.

Distribution and origin of substance P- and neuropeptide Y-immunoreactive nerves in the guinea-pig heart

SummaryThe localization and origin of substance P (SP)-, neuropeptide Y (NPY)-, and noradrenaline/tyrosine hydroxylase (NA/TH)-immunoreactive (IR) nerves in the guinea-pig heart were investigated by means of immunohistochemistry; quantitative analysis was performed by radioimmunoassay (NPY) and high performance liquid chromatography (NA). Both untreated animals and animals subjected to stellatectomy, combined stellatectomy and local capsaicin pretreatment of the vagal nerves or systemic application of capsaicin were studied. A dense network of SP-IR nerves was observed in the right atrium in different locations: (1) around local cardiac ganglion cells, (2) close to blood vessels, (3) within the myocardium, and (4) close to and within peri and endocardium.A moderately dense SP-innervation, mainly related to blood vessels, was found in the ventricles. Very dense networks of NPY and TH-IR nerve fibers with an overlapping distributional pattern around blood vessels and in the myocardium were seen in both the atria and the ventricles. In addition, some cell bodies in local cardiac ganglia were NPY-IR. Bilateral stellatectomy resulted in a reduction of SP-IR in the right atrium (55% of control), which was more pronounced after additional capsaicin pretreatment of the vagal nerves (44% of control).In the left ventricle no significant depletion of SP-IR was seen by either stellatectomy or combined stellatectomy and capsaicin treatment of the vagal nerves. It was not possible to establish any defined target areas within the heart for vagal or spinal SP-IR afferents by use of immunohistochemical methods. Systemic capsaicin treatment caused a total loss of SP-IR nerves in the heart. After bilateral stellatectomy the levels of NPY-IR and NA were reduced to about 10% of control in both the right atrium and left ventricle. In accordance, NPY and TH-IR nerves were also almost totally absent in the heart after bilateral stellatectomy.

Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat

Using the indirect immunohistochemical technique with antisera to cholecystokinin and to substance P, the spinal dorsal horn and dorsal root ganglia of normal and colchicine-treated rats were studied. In the spinal cord a similar distribution of substance P- and cholecystokinin-positive networks in the superficial layers of the dorsal horn was observed. In the dorsal root ganglia several cholecystokinin and substance P immunoreactive cell bodies were seen in colchicine-treated rats. After elution and restaining for substance P, of sections previously stained for cholecystokinin, it was found that all cholecystokinin-positive cells also contained substance P-like immunoreactivity and vice versa.

Decreased Survival of Experimental Critical Flaps in Rats after Sensory Denervation with Capsaicin

The role of capsaicin-sensitive primary sensory neurons on the survival of experimental critical flaps was studied in the rat. Pretreatment with capsaicin, which depletes neuropeptide transmitter content from primary sensory neurons, caused a dramatic decrease in flap survival area compared to normal animals. In contrast, pretreatment with reserpine, which depletes catecholamines from adrenergic neurons, including the sympathetic post-ganglionic fibers, resulted in a significant increase in the survival area. It was concluded that both capsaicin-sensitive primary sensory neurons and sympathetic postganglionic adrenergic neurons play a role in systemic vascular regulation and that intact primary sensory neurons are of importance for the survival of ischemic tissue.

Localization of substance P-immunoreactive nerve fibers in the human digital skin

Substance P-immunoreactive nerve endings were localized in human digital skin by the use of indirect immunohistochemical technique. It was found that substance P-like immunoreactivity was present in free nerve endings in the dermal papillae and in the epidermis. Some Meissners corpuscles also contained substance P positive nerve endings. Furthermore, substance P-immunoreactive nerves were localized in close connection to sweat gland ducts and blood vessels. The functional significance of these findings was discussed with regard to pain mediation and inflammatory response.

Dynorphin-immunoreactive neurons in the autonomic nervous system

The distribution of dynorphin-like immunoreactivity in the autonomic nervous system of the rat and guinea-pig was investigated using an antiserum raised against dynorphin-(1-17). Dynorphin-like immunoreactivity was observed in fiber networks in the prevertebral sympathetic ganglia, in fibers in the enteric plexa , circular muscle layer and a few in the lamina muscularis of the mucosa of the gastrointestinal tract. After colchicine treatment dynorphin-immunoreactive cell bodies were observed both in the myenteric and submucous ganglia of the gut. The paravertebral ganglia contained occasional dynorphin-positive fibers and a few immunoreactive small intensely fluorescent cells. The distribution of the dynorphin-like immunoreactivity was compared to the distribution of enkephalin-like immunoreactivity, and it was found that in some areas there were similarities in the distribution patterns, although in other areas there were clear differences, indicating a non-identity of these two systems.