Robert Crayton

Axotomy-induced changes in the chemical coding pattern of colon projecting calbindin -positive neurons in the inferior mesenteric ganglia of the pig.

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

Proliferative Enteropathy (PE)—Induced Changes in the Calbindin-Immunoreactive (CB-IR) Neurons of Inferior Mesenteric Ganglion Supplying the Descending Colon in the Pig

A subpopulation of the pig inferior mesenteric ganglia (IMG) neurons projecting to the colon exhibit calbindin-like immunoreactivity. It is not known if there are any changes in the chemical coding patterns of these neurons during porcine proliferative enteropathy (PE). To answer this question, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PE; n = 3) and a group of uninfected controls (C; n = 3) were compared. The retrograde tracer fast blue (FB) was injected into the descending colons of all animals and then tissue comprising IMGs from both groups was processed for double-labeling immunofluorescence with calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase, Leu-enkephalin, substance P, vesicular acetylcholine transporter, galanin, or pituitary adenylate cyclase-activating polypeptide. Immunohistochemistry revealed changes in the chemical coding pattern of calbindin-immunoreactive neurons in the inferior mesenteric ganglia of the pig. In control animals, FB/CB-positive neurons were immunoreactive to TH, NPY, SOM, and VIP. In the experimental group, TH-expressing neurons were unaffected, NPY-expressing neurons were increased, whereas the number of neurons immunoreactive to SOM or VIP was reduced. Changes in chemical coding of CB neurons during PE may play an important role in adaptation of these IMG cells under pathological conditions.

Urethral distension as a novel method to simulate sphincter insufficiency in the porcine animal model

Objectives:  To describe a novel animal model of intrinsic sphincter deficiency.

The Effect of Endoscopic Administration of Autologous Porcine Muscle-derived Cells Into the Urethral Sphincter

OBJECTIVE To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. METHODS This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. RESULTS The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. CONCLUSION The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

Chemical coding of zinc-enriched neurons in the intramural ganglia of the porcine jejunum

Zinc ions in the synaptic vesicles of zinc-enriched neurons (ZEN) seem to have an important role in normal physiological and pathophysiological processes in target organ innervation. The factor directly responsible for the transport of zinc ions into synaptic vesicles is zinc transporter 3 (ZnT3), a member of the divalent cation zinc transporters and an excellent marker of ZEN neurons. As data concerning the existence of ZEN neurons in the small intestine is lacking, this study was designed to disclose the presence and neurochemical coding of such neurons in the porcine jejunum. Cryostat sections (10 mμ thick) of porcine jejunum were processed for routine double- and triple-immunofluorescence labeling for ZnT3 in various combinations with immunolabeling for other neurochemicals including pan-neuronal marker (PGP9.5), substance P (SP), somatostatin (SOM), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin (LENK), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), galanin (GAL), and calcitonin-gene related peptide (CGRP). Immunohistochemistry revealed that approximately 39%, 49%, and 45% of all PGP9.5- positive neurons in the jejunal myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, were simultaneously ZnT3+. The majority of ZnT3+ neurons in all plexuses were also VAChT-positive. Both VAChT-positive and VAChT-negative ZnT3+ neurons co-expressed a variety of active substances with diverse patterns of co-localization depending on the plexus studied. In the MP, the largest populations among both VAChT-positive and VAChT-negative ZnT3+ neurons were NOS-positive cells. In the OSP and ISP, substantial subpopulations of ZnT3+ neurons were VAChT-positive cells co-expressing SOM and GAL, respectively. The broad-spectrum of active substances that co-localize with the ZnT3+ neurons in the porcine jejunum suggests that ZnT3 takes part in the regulation of various processes in the gut, both in normal physiological and during pathophysiological processes.

Inflammation-Induced Changes in the Chemical Coding Pattern of Colon-Projecting Neurons in the Inferior Mesenteric Ganglia of the Pig

The present study examines the chemical coding of the inferior mesenteric ganglia after chemically induced colitis in the pig animal model. In all animals (n = 6), a median laparotomy was performed under anesthesia, and the Fast Blue retrograde tracer was injected into the descending colon wall. In experimental animals (n = 3), the thick descending colon were injected with formalin solution to induce inflammation. The animals were euthanized and the inferior mesenteric ganglion was harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL). Immunohistochemistry revealed significant changes in the chemical coding pattern of inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, LENK, CB, and NOS. In the experimental group, TH, NPY, SOM, VIP, and LENK expressing neurons were reduced, whereas the number of neurons immunoreactive to CB, NOS, and GAL were increased. The increase of so-called neuroprotective neuropeptides suggests that the changes in the chemical coding of inferior mesenteric ganglion neurons reflect adaption under pathological conditions to promote their own survival.

Innervation pattern of polycystic ovaries in the women.

The aim of the present study was to determine the changes in both the distribution pattern and density of nerve fibers containing dopamine β-hydroxylase (DβH), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SOM), galanin (GAL) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the human polycystic ovaries. In the polycystic ovaries, when compared to the immunoreactions pattern observed in the control gonads, following changes were revealed: (1) an increase in the number of DβH-, VAChT-, VIP- or GAL-immunoreactive (IR) nerve fibers within the stroma as well as in the number of DβH-IR fibers near primordial follicles and medullar veins and venules; (2) a reduction in the number of nerve fibers containing nNOS, CGRP, SOM, PACAP within the stroma and in the numbers of CGRP-IR fibers around arteries; (3) an appearance of SP- and GAL-IR fibers around medullar and cortical arteries, arterioles, veins and venules, with except of GAL-IR fibers supplying medullar veins; and (4) the lack of nNOS-IR nerve fibers near primordial follicles and VIP-IR nerves around medullar arteries and arterioles. In conclusion, our results suggest that the changes in the innervation pattern of the polycystic ovaries in human may play an important role in the pathogenesis and/or course of this disorder.

Exogenously Administered Opioids Contract the Female Rat Intrinsic Urethral Sphincter In Vivo

Previous studies have reported immunoreactive opioid nerve fibers in the detrusor and lower urinary tract sphincters. However, there is a paucity of in vivo studies demonstrating the direct effect of endogenous opioids in these structures. In the present study, we investigated the contractile actions of intra‐arterially administered exogenous Dynorphin‐A, Met‐enkephalin, Leu‐enkephalin, morphine, and the opioid antagonist naltrexone on the female rat intrinsic urethral sphincter in vivo.

Exogenously Administered Bombesin and Gastrin Releasing Peptide Contract the Female Rat Urethra In Vivo and In Vitro

Bombesin (BOM) and gastrin releasing peptide (GRP) have been located to the lower urinary tract (LUT). However, there is a paucity of data demonstrating the impact of these endogenous peptides.