Jerzy Kaleczyc

The distribution and co-localization of immunoreactivity to nitric oxide synthase, vasoactive intestinal polypeptide and substance P within nerve fibres supplying bovine and porcine female genital organs.

The distribution of nitric oxide synthase-immunoreactive (NOS-IR) axons and their relationship to structures immunoreactive to vasoactive intestinal polypeptide (VIP), substance P (SP) and tyrosine hydroxylase (TH) were studied by means of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) technique or double-labelling immunofluorescence in the genital organs of cow and pig. Relevant neurons were also investigated in the pig. NOS-containing neural structures were TH-immunonegative in bovine or porcine genital organs, or in the studied ganglia. In the bovine ovary, NOS-IR nerves were neither VIP-IR nor SP-IR, whereas in the pig, most NOS-containing axons were also VIP-IR. The oviduct was supplied by single NOS/VIP- or NOS/SP-containing nerves, whereas in the uterus, NOS-IR axons were moderate in number, often being immunoreactive for VIP or SP. Numerous NOS/VIP-IR and NOS/SP-IR nerves were found in the vagina of both species. In all tissues studied, NOS-IR axons were mainly related to vascular smooth muscle. Most of the neurons of the paracervical ganglia and some neurons in dorsal root ganglia exhibited strong NOS activity. Only single neurons in sympathetic ganglia were NADPH-d-positive. Most nitrergic neurons in the autonomic ganglia were VIP-IR but SP-immunonegative. The sensory neurons were mostly NOS/SP-IR, whereas only single neurons co-expressed NOS and VIP immunoreactivity.

Origin and neurochemical characteristics of nerve fibres supplying the mammalian vas deferens

The present paper deals with the origin and neurochemical characteristics of autonomic postganglionic and sensory nerve fibres supplying the mammalian vas deferens. The vas deferens is innervated by postganglionic nerve fibres originating primarily from neurons in pelvic ganglia and, to a lesser extent, from neurons in the inferior mesenteric ganglion and sympathetic chain ganglia as well as by sensory nerve fibres arising from dorsal root ganglia. Three major populations of nerve terminals innervating the organ can be distinguished: (1) noradrenergic fibres; (2) cholinergic fibres containing vasoactive intestinal polypeptide, neuropeptide Y, nitric oxide synthase, and (in the pig) somatostatin, supplying particularly the lamina propria; and (3) non‐noradrenergic, presumably sensory fibres, containing calcitonin gene‐related peptide and/or substance P. The population of noradrenergic nerves is the most common. In the pig, it can be divided into three subpopulations: a somatostatin‐containing, a Leu‐enkephalin‐containing and a subpopulation immunonegative to these peptides, in descending order of magnitude. In the rat, guinea‐pig, and man, NPY seems to be the most common peptide occurring in the noradrenergic axons. In the pig, coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non‐noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the mammalian vas deferens and its blood vessels is discussed. Microsc. Res. Tech. 42:409–422, 1998.

Distribution and immunohistochemical characteristics of neurons in the porcine caudal mesenteric ganglion projecting to the vas deferens and seminal vesicle

Combined retrograde tracing (using fluorescent tracer Fast Blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons in the porcine caudal mesenteric ganglion projecting to the vas deferens and seminal vesicle. The distribution and immunohistochemical properties of neurons projecting to both organs were similar. As revealed by retrograde tracing, Fast Blue-positive neurons were located within the left and right ganglia, with a distinct predominance in the ipsilateral one. In the ipsilateral ganglion, the majority of the neurons were located caudally, along the dorso-lateral ganglionic border, suggesting a somatotopic organization of the ganglion. Immunohistochemistry revealed four populations of retrogradely labelled neurons (from the largest to the smaller one): tyrosine hydroxylase-positive/neuropeptide Y-negative (TH+/NPY-), TH+/NPY+, TH-/NPY-, TH-/NPY+. With respect to their surrounding nerve fibres, two subpopulations of the dye-labelled neurons could be distinguished. The small one consisted of solitary neurons receiving a strong calcitonin gene-related peptide- and Leu5-enkephalin-, and a less intense vasoactive intestinal peptide-immunoreactive innervation. The remaining neurons were poorly supplied by singular nerve fibres containing some of the investigated peptides. We conclude that the caudal mesenteric ganglion should be considered as a prominent source of adrenergic and/or NPY-positive innervation for the porcine male reproductive tract.

Immunohistochemical properties of nerve fibres supplying accessory male genital glands in the pig. A colocalisation study.

Abstract Immunohistochemical studies have been performed to investigate the occurrence and coexistence of two catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase, and several neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, Leu5-enkephalin, somatostatin, calcitonin gene-related peptide and substance P, in nerve fibres supplying porcine accessory genital glands, the seminal vesicles, prostate (body and the disseminated part) and bulbourethral glands. Three major populations of nerve fibres supplying non-vascular elements of the glands have been distinguished (from the largest to the smallest one): (1) noradrenergic fibres, the majority of which contain Leu5-enkephalin, neuropeptide Y or, to a lesser extent, somatostatin, (2) non-noradrenergic, putative cholinergic fibres containing vasoactive intestinal polypeptide, neuropeptide Y and/or somatostatin and, (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. Whilst the coexistence patterns within nerves supplying particular glands are similar, the density of innervation varies between the organs. The innervation of the seminal vesicles and prostatic body is more developed than that of the disseminated part of the prostate and bulbourethral glands. The majority of noradrenergic fibres related to blood vessels contain neuropeptide Y only, while the non-noradrenergic nerves contain mainly vasoactive intestinal polypeptide. The possible function and origin of particular nerve fibre populations are discussed.

Innervation of the fibro-elastic type of the penis: an immunohistochemical study in the male pig

The occurrence and colocalization of several biologically active neuropeptides, catecholamine-, acetylcholine- or nitric oxide-synthesizing enzymes-tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (D beta H), choline acetyl-transferase (ChAT) and nitric oxide synthase (NOS I), respectively, as well as the vesicular acetylcholine transporter (VAChT) were investigated in the penile glans (GP), corpus and crura (CP), as well as in the retractor penis muscle (RPM) of juvenile and adult boars. Immunohistochemistry revealed that nerves immunoreactive (IR) to TH, D beta H, vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were the most numerous, followed (in decreasing order of density) by nerves IR to NOS, neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), Leu5-enkephalin (LENK) and ChAT/VAChT. The CP contained the largest number of nerve fibres followed by the RPM, GP and corpus. Enzyme/peptide-containing nerves were associated with both the vascular and non-vascular penile structures. However, differences existed for their density and intrapenile distribution. Nerve terminals IR for different combinations of VIP, GAL or SOM were more frequent than those IR for NOS or CGRP in the non-vascular penile structures while the vasculature and the RPM received a prominent TH/D beta H-, VIP-, SOM- or NOS-IR nerve input. The present data indicate that the porcine penis receives nerve fibres that exhibit diverse chemical codes and that differences in the chemical coding of the nerve fibres may depend on their penile target-structure.

Immunohistochemical characteristics of nerve fibres supplying the porcine vas deferens. A colocalisation study

Abstract Double-labelling immunofluorescence was used to investigate the coexistence of the catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase and several neuropeptides including neuropeptide Y, vasoactive intestinal polypeptide, Leu5-enkephalin, somatostatin, calcitonin gene-related peptide and substance P in nerve fibres supplying the vas deferens in juvenile and adult pigs. The study has revealed three major populations of nerve terminals innervating the organ: (1) noradrenergic fibres; (2) non-noradrenergic (putative cholinergic) fibres containing vasoactive intestinal polypeptide, neuropeptide Y and somatostatin, supplying almost exclusively the lamina propria; and (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. The population of noradrenergic nerves can be divided into three subpopulations: a somatostatin-containing, a Leu5-enkephalin-containing and a subpopulation immunonegative to the peptides investigated, in descending order of magnitude. Coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non-noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the porcine vas deferens and its blood vessels is discussed.

Peptidergic innervation of the bovine vagina and uterus

The distribution of neuropeptide Y, substance P, vasoactive intestinal polypeptide, Leu5-enkephalin, bombesin/gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin, cholecystokinin and catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase was studied immunohistochemically in nerve fibres supplying the bovine vagina and uterus. The nerves containing tyrosine hydroxylase or dopamine-beta-hydroxylase and neuropeptide Y-immunoreactivity were particularly numerous in both organs. Substance P, vasoactive intestinal polypeptide and Leu5-enkephalin-containing nerves were less numerous whereas somatostatin and calcitonin gene-related peptide-immunoreactive nerves occurred occasionally. Bombesin/gastrin-releasing peptide and cholecystokinin immunoreactivities were not present in nervous fibers of the bovine uterus and vagina. Generally, the immunoreactive nerve terminals, fibers, networks or nerve bundles were present below the serous membrane, between smooth muscle cells of muscular layers, around blood vessels, in the submucosal layer and below the luminal epithelium of the uterus and cervix.

Existence and co-existence of vasoactive substances in nerve fibres supplying the abdomino-pelvic arterial tree of the female pig and cow

The occurrence and co-localization of several presumed vasoactive neuropeptides, serotonin, and catecholamine-synthesising enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (D beta H) and phenylethanolamine-N-methyltransferase (PNMT)--were investigated in perivascular nerves supplying the systemic and distributing arteries of the abdomino-pelvic arterial tree of the female pig and certain arteries supplying female reproductive organs in the cow. As revealed by single immunofluorescence, perivascular axons immunoreactive for TH, D beta H, neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and Leu-enkephalin (LENK) occurred in both species examined, whereas galanin-immunoreactive (GAL-IR) nerve fibres were found exclusively in the pig. PNMT-, serotonin-, dynorphin A-, alpha-neoendorphin-, bombesin- or cholecystokinin-IR nerve terminals were not observed. The following classes of perivascular nerve fibres might be distinguished in the present study: 1) noradrenergic (i.e. TH/D beta H-IR), 2) NPY-, 3) GAL- (pig only), 4) LENK-, 5) VIP-, 6) SP-, 7) VIP/NPY-, 8) SP/CGRP-, 9) SP/GAL- (pig only), 10) SP/VIP- (cow only), 11) TH/D beta H/NPY- and 12) TH/D beta H/NPY/LENK-IR. Distinct differences in the distribution of LENK- and SP-IR axons around particular parts of the studied arterial tree in individual species were also observed. The present data indicate that the abdomino-pelvic arterial tree of the pig and cow receive perivascular nerve fibres that exhibit diverse chemical codes, and that different chemical coding of perivascular nerve fibres in individual species may depend on the target organ of the particular artery.

Somatostatin, substance P and calcitonin gene-related peptide-positive intramural nerve structures of the human large intestine affected by carcinoma

The aim of this study was to investigate the arrangement and chemical coding of enteric nerve structures in the human large intestine affected by cancer. Tissue samples comprising all layers of the intestinal wall were collected during surgery form both morphologically unchanged and pathologically altered segments of the intestine (n=15), and fixed by immersion in buffered paraformaldehyde solution. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP). The microscopic observations revealed distinct morphological differences in the enteric nerve system structure between the region adjacent to the cancer invaded area and the intact part of the intestine. In general, infiltration of the cancer tissue resulted in the gradual (depending on the grade of invasion) first decomposition and reduction to final partial or complete destruction and absence of the neuronal elements. A comparative analysis of immunohistochemically labeled sections (from the unchanged and pathologically altered areas) revealed a statistically significant decrease in the number of CGRP-positive neurons and nerve fibres in both submucous and myenteric plexuses in the transitional zone between morphologically unchanged and cancer-invaded areas. In this zone, a decrease was also observed in the density of SP-positive nerve fibres in all intramural plexuses. Conversely, the investigations demonstrated statistically insignificant differences in number of SP- and SOM-positive neurons and a similar density of SOM-positive nerve fibres in the plexuses of the intact and pathologically changed areas. The differentiation between the potential adaptive changes in ENS or destruction of its elements by cancer invasion should be a subject of further investigations.

Immunohistochemical Localization of Galanin in Bovine Reproductive Organs

The vagina, uterus and oviduct were shown to receive galanin immunoreactive (GAL‐IR) nerve fibres, the number of which varied between particular organs. In the ovary, GAL‐IR nerves were absent. A small number of these nerves were located in the layers of the oviduct. A moderate number of GAL‐IR nerves were situated in the body and uterine horns, whereas the uterine cervix and vagina wall contained a large number of GAL‐IR nerve fibres, evenly distributed throughout particular membranes of the organs. GAL‐IR nerves were found to contain, simultaneously, either vasoactive intestinal polypeptide (VIP), substance P (SP) or Leu5‐enkephalin (ENK). Many of the GAL‐IR nerves contained tyrosine hydroxylase (TH). A group of GAL‐IR nerves that did not possess immunoreactivity to VIP, SP, ENK or TH was also observed.