Complementary mechanisms of modulation of spontaneous phasic contractions by the gaseous signalling molecules NO, H2S, HNO and the polysulfide Na2S3 in the rat colon

Objectives Reactive oxygen and nitrogen species may be produced during inflammation leading to the formation of NO, H2S or HNO. Enzymes such as iNOS, CSE and CBS might also be responsible for polysulfide production. Since these signalling molecules might have an impact on colonic motility, the aim of this study was to compare their effect on rat colonic slow phasic contractions (SPC). Methods Organ bath measurements with strips obtained from rat proximal colon were performed using the polysulfide Na2S3, sodium nitroprusside (NaNP), sodium hydrogen sulfide (NaHS), Angeli’s salt as NO, H2S, and HNO donors, respectively. TTX (1 µM) was used to block neuronal activity. Results All four molecules, concentration-dependently, inhibited the amplitude and frequency of SPC both in the circular and longitudinal muscle layer. The relative potency was NaNP>Angeli’s salt>NaHS>Na2S3. The inhibitory response induced by NaNP (1 µM) and Angeli’s salt (50 µM) was reversed by ODQ (10 µM) whereas the inhibitory effect of NaHS (1 mM) was reversed by apamin (1 µM) and glibenclamide (10 µM). Na2S3 (1 mM) response was partially reversed by apamin (1 µM) and glibenclamide (10 µM). High concentrations of Na2S3 caused an increase in tone. Low concentrations of NaHS or Na2S3 did not potentiate NaNP responses. Conclusions All signalling molecules inhibit SPC in both muscle layers. The effect is independent of neural activity and involves guanylyl cyclase (NO and HNO) and SKCa and KATP channels (NaHS or Na2S3). Other pathways might also be involved in Na2S3 responses. Accordingly, complementary mechanisms of inhibition might be attributable to these signalling molecules.

Persistent Herpes Simplex Virus Type 1 Infection of Enteric Neurons Triggers CD8+ T Cell Response and Gastrointestinal Neuromuscular Dysfunction

Behind the central nervous system, neurotropic viruses can reach and persist even in the enteric nervous system (ENS), the neuronal network embedded in the gut wall. We recently reported that immediately following orogastric (OG) administration, Herpes simplex virus (HSV)-1 infects murine enteric neurons and recruits mononuclear cells in the myenteric plexus. In the current work, we took those findings a step forward by investigating the persistence of HSV-1 in the ENS and the local adaptive immune responses against HSV-1 that might contribute to neuronal damage in an animal model. Our study demonstrated specific viral RNA transcripts and proteins in the longitudinal muscle layer containing the myenteric plexus (LMMP) up to 10 weeks post HSV-1 infection. CD3+CD8+INFγ+ lymphocytes skewed towards HSV-1 antigens infiltrated the myenteric ganglia starting from the 6th week of infection and persist up to 10 weeks post-OG HSV-1 inoculation. CD3+CD8+ cells isolated from the LMMP of the infected mice recognized HSV-1 antigens expressed by infected enteric neurons. In vivo, infiltrating activated lymphocytes were involved in controlling viral replication and intestinal neuromuscular dysfunction. Indeed, by depleting the CD8+ cells by administering specific monoclonal antibody we observed a partial amelioration of intestinal dysmotility in HSV-1 infected mice but increased expression of viral genes. Our findings demonstrate that HSV-1 persistently infects enteric neurons that in turn express viral antigens, leading them to recruit activated CD3+CD8+ lymphocytes. The T-cell responses toward HSV-1 antigens persistently expressed in enteric neurons can alter the integrity of the ENS predisposing to neuromuscular dysfunction.

Evidence of enteric angiopathy and neuromuscular hypoxia in patients with mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by thymidine phosphorylase (TP) enzyme defect. Since gastrointestinal changes do not revert in patients undergone TP replacement therapy, one can postulate that other unexplored mechanisms contribute to MNGIE pathophysiology. Hence, we focused on the local TP angiogenic potential that has never been considered in MNGIE. In this study, we investigated the enteric submucosal microvasculature and the effect of hypoxia on fibrosis and enteric neurons density in jejunal full-thickness biopsies collected from MNGIE patients. Orcein staining was used to count blood vessels based on their size. Fibrosis was assessed using the Sirius Red and Fast Green method. Hypoxia and neoangiogenesis were determined via hypoxia-inducible-factor-1a (HIF-1a) and vascular endothelial cell growth factor (VEGF) protein expression, respectively. Neuron specific enolase was used to label enteric neurons. Compared to controls, MNGIE patients showed a decreased area of vascular tissue, but a two-fold increase of submucosal vessels/mm2 with increased small size and decreased medium and large size vessels. VEGF positive vessels, fibrosis index and HIF-1a protein expression were increased, whereas there was a diminished thickness of the longitudinal muscle layer with an increased inter-ganglionic distance and reduced number of myenteric neurons. We demonstrated the occurrence of an angiopathy in the GI tract of MNGIE patients. Neoangiogenetic changes, as detected by the abundance of small size vessels in the jejunal submucosa, along with hypoxia provide a morphological basis to explain neuromuscular alterations, vasculature breakdown and ischemic abnormalities in MNGIE.

Diversification of leech proboscis structure according to prey ingestion behavior

Background Adaptive radiation is a phenomenon in which various organs are diversified morphologically or functionally as animals adapt to environmental inputs such as diet and circumstance. Although previous studies have addressed changes caused by various external pressures, the evidence for variation in invertebrates is not well known. Leeches comprise a carnivorous or ectoparasitic group of animals that feed on a wide range of prey. They exhibit a corresponding variety of ingestion behaviors and morphological diversity of mouthparts and gut specializations. However, research on the diversity of ingestion behaviors and the internal structure of feeding organs in leeches is little known. In this study, we use histological analyses, fluorescent labeling and immunohistochemistry to reveal the detailed proboscis structure in the family Glossiphoniidae, while also suggesting the diversification of proboscises.Results We identified the feeding behavior of rhynchobdellid leeches, which have the proboscises. Alboglossiphonia sp. swallows prey whole using its proboscis, whereas other leeches exhibit typical fluid-sucking behavior. Glossiphoniid leeches exhibit fluid ingestion behavior along with clear arrangement of longitudinal muscles, circular muscles surrounding the lumen, and radial muscles, while Alboglossiphonia sp., which displays macrophagous ingestion like salifid Barbronia sp., has a partial circular muscle distribution and spacious lumen that extends to longitudinal muscle layer. To address whether the different feeding behaviors are intrinsic, we investigated the behavioral patterns and muscle arrangements in the earlier developmental stage of glossiphoniid leeches. Juvenile Glossiphoniidae including the Alboglossiphonia sp. exhibit the fluid ingestion behavior and have the proboscis with the compartmentalized muscle layers.Conclusions Genetic, morphological and behavioral differences between juvenile and adult stages of Alboglossiphonia sp. suggest their adult feeding biology has diverged from ancestral glossiphoniid leeches, while retaining developmental vestiges of the typical juvenile feeding morphology currently observed across Glossiphoniidae. This study provides the characteristics of leeches with specific ingestion behaviors, and a comparison of structural differences that serves as the first evidence of the proboscis diversification.

Redescription of Cerebratulus marginatus auct. (Nemertea: Pilidiophora) from Hokkaido, Japan, as a new species

The heteronemertean Cerebratulus orochi sp. nov. is described based on material collected intertidally at a muddy beach in Akkeshi, northern Japan. For the last 80 years, the species has been confused with Cerebratulus marginatus Renier, 1804; the latter was originally described from the Adriatic and once believed to occur in many places in the northern hemisphere including Japan. Cerebratulus orochi sp. nov. is morphologically different from all the congeners including C. marginatus by the following combination of characters: several layers of diagonal-muscle meshwork coated with connective tissue, proximo-distally distributed in cross section from the distal portion of the body-wall outer longitudinal muscle layer to the cutis-gland zone throughout the anterior portion of the body from the precerebral to the foregut regions; the cephalic vascular system consisting of lateral and mid-dorsal vessels; and the sub-rhynchocoelic vessel possessing a pair of antero-lateral diverticula before the former forks posteriorly into a pair of lower lateral vessels in the post-cerebral, pre-oral region. Previous records of C. marginatus from Japanese waters are no longer considered to be substantiated. Multi-locus phylogenetic analyses based on the mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI), as well as the nuclear 18S rRNA, 28S rRNA, and histone H3 genes among heteronemerteans comprising the “Cerebratulus clade” indicated that C. orochi sp. nov. was closely related to C. cf. marginatus from the US Pacific coast. A MegaBLAST search at the NCBI website with the 16S rRNA gene sequence from C. orochi sp. nov. followed by a couple of species delimitation analyses suggests that larvae of the species are also distributed in Vostok Bay, Far East Russia.

MORPHOLOGICAL CHANGES IN THE MUSCLE LAYERS OF SMALL INTESTINE IN THE SHORT BOWEL SYNDROME IN EXPERIMENT

Introduction. Patients with the short bowel syndrome often have intestinal dilatation which impairs intestinal functions. Changes in the muscle intestinal layers, which cause this condition, are not studied well yet. Purpose. To study the role of small intestine muscle layers in the intestinal adaptation and dilatation in the short bowel syndrome. Materials and methods. 22 rats were taken into the experimental trial; short bowel syndrome was modelled in 12 of them; 10 other rats which had only laparotomy were in the control group. The diameter and thickness of muscle layers in the small intestine and ileum were studied. Results. In the short bowel syndrome, one can observe a significant dilatation of the small intestine and ileum. Thickness of longitudinal and circular layers of the small intestine was significantly larger in rats with the short bowel syndrome in comparison to the control group. In the ileum, only the circular muscle layer was hypertrophied; there was no difference in the thickness of longitudinal muscle layer in rats with the short bowel syndrome and in rats from the control group. Conclusion. In rats with the short bowel syndrome, morphological changes occur not only in the mucous layer, but also in muscle layers of the small intestine what is manifested by the intestinal dilatation and hypertrophy of muscle layers. These changes are results of intestinal adaptation and are pathophysiological for the short bowel syndrome.

Morphology and microcirculatory structure of the urethra of the pig: Urologic surgical interest

The urethra of the sow can play an important role in the surgical revascularization or in the reconstructive surgery. However, the urethra can be easily injured if it possessed well architecture and vascularization. We investigated the morphological structure and vasculature features of the urethra to improve surgical results. Eight pelvises of sow were investigated. The block was cuts according to the segments of the urethra. They were made transparent according to the sagittal and transvers planes and examined trough Chinese ink agar injection. The adventia of both portions of the urethra of the sow (pelvic and perineal) were dependent on the collaterals of the internal iliac arteries and internal pudendal arteries. The veins of the periphery of the urethra derived from the muscle layers and the chorion and ran into a network in contact with arteries. The arteries of the striated muscle layer originated from the adventitial arteries of the anterior and lateral regions of the urethra. The chorion was depended on long vessels from the adventitious network and the terminal branches of the vessels of the inner longitudinal muscle layer. The arteries of the urethrovaginal septum were thin and came from the branches of the vaginal artery. The urethra of the sow was sheathed by the pelvic fascia lined with the elevated muscle of anus. They were well vascularized and can be safety exposed and preserved during experimental surgical dissection with careful drilling.Keys words: Sow urethra, morphology, structure, microvascularization.

First reported case of per anal endoscopic myectomy (PAEM): A novel endoscopic technique for resection of lesions with severe fibrosis in the rectum

Background and study aims A 54-year-old man was diagnosed with a rectal tumor extending through the submucosal layer. The patient refused surgery and therefore endoscopic submucosal dissection (ESD) was pursued. The lesion exhibited the muscle retraction sign. After dissecting circumferentially around the fibrotic area by double tunneling method, a myotomy was performed through the internal circular muscle layer, creating a plane of dissection between the internal circular muscle layer and the external longitudinal muscle layer, and a myectomy was completed.The pathologic specimen verified T1b grade 1 sprouting adenocarcinoma with 4350 µm invasion into the submucosa with negative resection margins.

Two Cases of Rectal Neuroendocrine Tumor Resection Combined with Dissection of the Circular Muscle Layer Using the Endoscopic Submucosal Dissection Technique

Generally, lesions of rectal neuroendocrine tumors (NETs) 10 mm or smaller are less malignant and are indicated for endoscopic therapy. However, the vertical margin may remain positive after conventional endoscopic mucosal resection (EMR) because NETs develop in a way similar to submucosal tumors (SMTs). The usefulness of EMR with a ligation device, which is modified EMR, and endoscopic submucosal dissection (ESD) was reported, but no standard treatment has been established. We encountered 2 patients in whom rectal NETs were completely resected by combined dissection and resection of the circular muscle layer using the ESD technique. Case 1 was an 8-mm NET of the lower rectum. Case 2 was NET of the lower rectum treated with additional resection for a positive vertical margin after EMR. In both cases, the circular muscle layer was dissected applying the conventional ESD technique, followed by en bloc resection while conserving the longitudinal muscle layer. No problems occurred in the postoperative course in either case. Rectal NETs are observed in the lower rectum in many cases, and it is less likely that intestinal perforation by endoscopic therapy causes peritonitis. The method employed in these cases, namely combined dissection and resection of the circular muscle layer using the ESD technique, can be performed relatively safely, and it is possible to ensure negativity of the vertical margin. In addition, it may also be useful for additional treatment of cases with a positive vertical margin after EMR.

Regulation and dysregulation of esophageal peristalsis by the integrated function of circular and longitudinal muscle layers in health and disease

Muscularis propria throughout the entire gastrointestinal tract including the esophagus is comprised of circular and longitudinal muscle layers. Based on the studies conducted in the colon and the small intestine, for more than a century, it has been debated whether the two muscle layers contract synchronously or reciprocally during the ascending contraction and descending relaxation of the peristaltic reflex. Recent studies in the esophagus and colon prove that the two muscle layers indeed contract and relax together in almost perfect synchrony during ascending contraction and descending relaxation of the peristaltic reflex, respectively. Studies in patients with various types of esophageal motor disorders reveal temporal disassociation between the circular and longitudinal muscle layers. We suggest that the discoordination between the two muscle layers plays a role in the genesis of esophageal symptoms, i.e., dysphagia and esophageal pain. Certain pathologies may selectively target one and not the other muscle layer, e.g., in eosinophilic esophagitis there is a selective dysfunction of the longitudinal muscle layer. In achalasia esophagus, swallows are accompanied by the strong contraction of the longitudinal muscle without circular muscle contraction. The possibility that the discoordination between two muscle layers plays a role in the genesis of esophageal symptoms, i.e., dysphagia and esophageal pain are discussed. The purpose of this review is to summarize the regulation and dysregulation of peristalsis by the coordinated and discoordinated function of circular and longitudinal muscle layers in health and diseased states.