Waldemar Sienkiewicz

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.

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.

Has Active Immunization Against Gonadotrophin‐Releasing Hormone any Effect on Testis Innervation in the Pig? – An Immunohistochemical Study

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin‐releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DβH), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), synaptosome‐associated protein of 25 kDa (SNAP‐25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH‐diaphorase histochemical technique or the Mayer counter‐staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non‐treated animals, only single TH‐immunoreactive (TH‐IR) nerve fibres were observed. However, many DβH‐IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP‐IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP‐25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH‐receptors and NADPH‐diaphorase. In the testes of immunized animals, only single TH‐IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH‐IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. DβH‐IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP‐IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP‐IR nerves were found between seminiferous tubules. Numerous NPY‐IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP‐25‐IR and PGP 9.5‐IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH‐diaphorase.

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.

Distribution of catecholamine-synthesizing enzymes and some neuropeptides in the median eminence-arcuate nucleus complex (MEARC) of the immature female pig.

The presence of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (D beta H) and some neuropeptides, including neuropeptide Y (NPY), Leu5-enkephalin (LENK), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL) and somatostatin (SOM) was investigated in nerve fibres and perikarya of the median eminence-arcuate nucleus complex (MEARC) of the sexually immature female pigs by means of the immunohistochemical avidin-biotin complex method. Although immunoreactivities to all the studied substances were found in nerve fibres of the porcine MEARC, there were differences in the distribution and density of particular subsets of nerve fibres within the complex. While loose D beta H-immunoreactive (D beta H-IR) and dense TH-, NPY- and VIP-IR nerve meshworks occurred predominantly in the internal layer of the MEARC, nerve fibres immunoreactive to TH, CGRP, SOM, SP and LENK were more numerous in the external than in the internal layer of the median eminence (ME). Numerous TH-, D beta H-, NPY-, VIP-, SP- and CGRP-IR perivascular nerve fibres were also observed within both layers of the median eminence. There were also differences in the distribution of a particular subset of neurons within the porcine MEARC: NPY-, VIP-, GAL-, SP- and TH-IR (but not D beta H-IR) perikarya were found in the arcuate nucleus, while in the median eminence only subpopulations of NPY-, VIP and GAL-IR neurons were observed.

Efficacy of lateral- versus medial-approach hip joint capsule denervation as surgical treatments of the hip joint pain; a neuronal tract tracing study in the sheep

Objective To evaluate efficacy of denervation of the of the hip joint capsule (HJC), as a treatment of hip joint pain. Specifically, we tested the hypothesis that HJC denervation will significantly reduce the number of sensory neurons innervating the capsule. Study design Denervation of the HJC from a medial or lateral approach was followed by retrograde tracing of sensory neurons innervating the capsule. Animals Twenty adult male sheep (30–40 kg of body weight; Polish merino breed) were used in the study. Methods The hip joint was denervated from medial (n = 5) or lateral (n = 5) surgical approaches. Immediately after denervation, the retrograde neural tract tracer Fast Blue (FB) was injected into the HJC. An additional ten animals (n = 5 for medial and n = 5 for lateral approach) received the same treatment without HJC denervation to provide the appropriate controls. Results Results of the study revealed that the vast majority of retrogradely labelled sensory neurons innervating the HJC originate from fifth lumbar to second sacral dorsal root ganglia. Both the medial and the lateral denervations significantly reduced the number of sensory neurons innervating the HJC (39.2% and 69.0% reduction respectively). Conclusions These results show that denervation of the HJC is an effective surgical procedure for reduction of the sensory neuronal input to the HJC. Moreover, the lateral approach was found to be significantly more effective for reducing sensory innervation as compared to the medial one.

Innervation of the chinchilla testis, epididymis, and vas deferens

Abstract The study was performed on six male chinchillas. The animals were anaesthetised with ether and the anaesthesia was deepened with nembuthal injected intraperitoneally. The chinchillas were then transcardially perfused with 0.4 L of 4% buffered paraformaldehyde, and testes, epididymides, and vasa deferentia were collected. The tunica albuginea from one testis from each chinchilla was stained as whole-mount preparation. The tissues were cut into 12 μm-thick cryostat sections, and processed for double-immunofluorescence method. In all organs studied, the most abundant nerve fibres were dopamine β hydroxylase positive (DβH+). Some of them contained neuropeptide Y (NPY). Sporadically NPY-positive-only nerve fibres were found. Single DβH+ nerve terminals contained also galanine. Small numbers of the nerve fibres supplying studied organs were stained for substance P (SP) and calitonin gene related peptide (CGRP). Almost all SP+ fibres were also CGRP+, whereas single CGRP+ nerves were SPimmunonegative. Some nerve terminals were immunoreactive to vesicular acetylcholine transporter and vasoactive intestinal peptide. The organs studied were innervated unevenly. The highest density of the nerves was found in the areas of the tunica albuginea adjacent to the mesorchial border of the testis and their number gradually decreased towards the free border of the gonad. None of the vascular tissue of the testicular parenchyma was free of the nerve fibres, except sporadically encountered DβH+ nerves which supply seminiferous tubules. Within the head of the epididymis a moderate number of nerve terminals were found, but in the body and tail of the organ the number of nerves gradually increased. The vas deferens was supplied with very numerous nerve fibres. There were no differences in the density of the innervation between the funicular and abdominal part of the vas deferens.