How Smooth Muscle Contractions Shape the Developing Enteric Nervous System

Neurons and glia of the enteric nervous system (ENS) are constantly subject to mechanical stress stemming from contractions of the gut wall or pressure of the bolus, both in adulthood and during embryonic development. Because it is known that mechanical forces can have long reaching effects on neural growth, we investigate here how contractions of the circular smooth muscle of the gut impact morphogenesis of the developing fetal ENS, in chicken and mouse embryos. We find that the number of enteric ganglia is fixed early in development and that subsequent ENS morphogenesis consists in the anisotropic expansion of a hexagonal honeycomb (chicken) or a square (mouse) lattice, without de-novo ganglion formation. We image the deformations of the ENS during spontaneous myogenic motility and show that circular smooth muscle contractile waves induce longitudinal strain on the ENS network; we rationalize this behavior by mechanical finite element modeling of the incompressible gut wall. We find that the longitudinal anisotropy of the ENS vanishes when contractile waves are suppressed in organ culture, showing that these contractile forces play a key role in sculpting the developing ENS. We conclude by summarizing different key events in the fetal development of the ENS and the role played by mechanics in the morphogenesis of this unique nerve network.

Study histological alterations of the effect of Deltamethrin on the skin of the earthworm

Our study aims to investigate all the histological changes that occur in skin tissues of the earthworms that exposure to deltamethrin at concentration (1:2000) g/L. our study is included two groups, the first group exposed to deltamethrin at a concentration (12.5) mg/L while the second group is included normal earthworms (control group). The soil samples were collected from local agricultural land randomly in the Daghara district in the city of Diwaniyah, then adding deltamethrin by distributed to the collected soil samples equally. The experimental group is consists of (20) samples wherever, which showed that the histological changes of the skin of the earthworm that exposure to deltamethrin (12.5) mg/L is included breaking in the cuticle layer, with clear degeneration in the longitudinal and circular smooth muscle fibers. Furthermore, the sub-dermal layer is very thin and destroyed with clear degeneration of the epidermal cells as well as showed slight congestion of the blood vessels under the nerve and a clear breakdown of the cerebral nerve ganglia, while the control group consists of (20) soil samples collected randomly, wherever, demonstrates that the tissue of the skin of the earthworm was naturally without changes, as a normal cuticle layer is observed, the epidermis layer is lined with normal epithelial cells, and the smooth muscle layer appears naturally arranged. In a conclusion, deltamethrin has histopathological effects on skin tissues of the earthworms that make are destroyed and leading to negative effects on the earthworm.

Role of PGE2 in colonic motility: PGE2 attenuates spontaneous contractions of circular smooth muscle via EP4 receptors in the rat colon

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called ‘giant contractions’ (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.

A132 SMOOTH MUSCLE BIOPSIES FROM POEM PROCEDURES FOR ACHALASIA SHOW LACK OF FUNCTIONAL INNERVATION

Background Idiopathic achalasia is a disease of the esophagus causing impaired peristalsis and absent lower esophageal sphincter relaxation. The etiopathogenesis of the disease is unclear but most histopathologic studies from surgical resections show an absence of nitric oxide-producing neurons in the myenteric plexus. The peroral endoscopic myotomy (POEM) procedure has evolved in the last decade to treat achalasia and has provided a unique way to sample diseased tissue from an otherwise inaccessible tissue compartment. Aims To study the effects of achalasia on innervation of smooth muscle and smooth muscle phenotype. Methods Patients with a diagnosis of achalasia based on high resolution manometry undergoing a POEM at Kingston general hospital were approached for enrollment between June 2017 to September 2018. Demographic information including age, symptom duration, previous treatments, and Eckhardt score was collected. Intraoperatively, biopsies of the circular smooth muscle (CSM) layer were taken at the proximal and distal extents of the myotomy and placed in formalin. Tissue was embedded in wax, sectioned and stained using immunocytochemistry (ICC) for neuronal, axonal and smooth muscle cell markers. This was compared to control tissue from patients undergoing gastroesophagectomy for adjacent malignancy. Results Control tissue from 3 separate esophagectomies were obtained. Samples were obtained from a total of 25 patients (13 males), with a median age of 50 (IQR 38–67). Most patients had Type 2 achalasia (19 [76%]) followed by Type 1 and 3 (2, [8%] respectively). Two cases had conflicting manometric findings. The median duration of symptoms was 3 years (IQR 1.5–10.5) and the median Eckhardt score was 6.5 (IQR 5–9). In sample tissues, no neuronal cell bodies were detected in the CSM layer. ICC for the axonal marker PGP9.5 showed that CSM of achalasia samples were almost completely devoid of axon structure, independent of the subtype of achalasia, compared to abundant axon presence in control tissues. In parallel, ICC showed that cholinergic (ChAT) or nitrergic (nNOS) axonal subtypes were absent in biopsy CSM while abundant in controls. CSM cells displayed hypertrophy with no detection of proliferation by ICC (KI67) or alterations in the phenotypic marker SM-22. Conclusions Preliminary results from advanced immunohistochemical techniques show the absence of functional innervation of the CSM layer of all patients with achalasia. This is characterized by a depleted excitatory and inhibitory axon population. Further studies are focused on defining differences in smooth muscle phenotype and the presence or absence of inflammatory cells within the CSM. Funding Agencies None

Smooth muscle contractility causes the gut to grow anisotropically

The intestine is the most anisotropically shaped organ, but, when grown in culture, embryonic intestinal stem cells form star- or sphere-shaped organoids. Here, we present evidence that spontaneous tonic and phasic contractions of the circular smooth muscle of the embryonic gut cause short-timescale elongation of the organ by a purely mechanical, self-squeezing effect. We present an innovative culture set-up to achieve embryonic gut growth in culture and demonstrate by three different methods (embryological, pharmacological and microsurgical) that gut elongational growth is compromised when smooth muscle contractions are inhibited. We conclude that the cumulated short-term mechanical deformations induced by circular smooth muscle lead to long-term anisotropic growth of the gut, thus demonstrating a self-consistent way by which the function of this organ (peristalsis) directs its shape (morphogenesis). Our model correctly predicts that longitudinal smooth muscle differentiation later in embryogenesis slows down elongation, and that several mice models with defective gut smooth muscle contractility also exhibit gut growth defects. We lay out a comprehensive scheme of forces acting on the gut during embryogenesis and of their role in the morphogenesis of this organ. This knowledge will help design efficient in vitro organ growth protocols and handle gut growth pathologies such as short bowel syndrome.

Changes in the Contractile Activity and Reactivity to 5-HT of Smooth Muscles of Rats Following Total Body Irradiation with Accelerated Electrons

Background: Besides its “classical” neurotransmitter function in the central and peripheral nervous systems, serotonin, or 5-hydroxytryptamine (5-HT) is also a local hormone in a number of tissues, including those of the GI tract. Radiation is known to be able to disrupt certain functions of the tract, modulated by 5-HT-signaling pathways, or the serotonin receptors themselves. Aim: The present investigation focused on clarifying the nature and extent of influence of an accelerated electron beam with energy of 9 MeV on the serotonergic mediation of healthy smooth muscle gastric tissue of rats following total body irradiation of the animals. Materials and methods: The study involved a control group and two experimental groups of animals exposed to 1 and 5 Gy, respectively, using Siemens Primus S/N 3561. Circular smooth muscle tissues were isolated from rats 1 hour and 18 hours after they were exposed to 1 and 5 Gy and also 5 days after irradiation from the rats that received a dose of 5 Gy in order to investigate the action of exogenous serotonin at increasing concentrations from 10-8 to 10-4 mol/l. The contractile reactivity of each group SM preparations was registered isometrically. Results: Electron beams with energy of 9 MeV did not damage the contractile apparatus of gastric SM of rats and had a stimulating effect on contractility resulting from rapidly developing processes (1 hour) or later occurring once (5 days). Conclusions: Difference was observed in the importance of the factors of received dose, lapse of time from irradiation to investigation of SM tissues, and exogenous 5-HT concentration for the changes in SM reactivity in serotonin-induced tonic and phasic responses.

KARAKTERISTIK MORFOLOGI DAN DISTRIBUSI KARBOHIDRAT NETRAL PADA UTERUS KELELAWAR BUAH (Pteropus vampyrus) ASAL PULAU TIMOR

Timorese fruit bat(Pteropusvampyrus)is the only fly mammalian with its unique behavior which hanging upside down inspite of its pregnancy. This research is aimed to reveal the morphology of the Timorese fruit bats and the distribution of neutral carbohydrate within this organ. Three uterus samples derived from three different Timorese fruit bats were used in the research.Both macroscopical and microscopical examinations using H&E and PAS methods were applied. Macroscopically, Timorese fruit bats showedsoft reddish white duplex uterus. Meanwhile microscopically, endometrium consisted of epithelial layer and lamina propria and was the place where simple tubular glands located. The epithelial layer comprised of simple cylindric secretory cells and ciliated cells. Neutral carbohydrate distribution was seen within this epithelial layer. Myometrium was a thick circular smooth muscle layer which consisted of smooth muscle separated by collagen and elastic fibre. Perimetrium was a visceral layer and consisted of mesothelial cells.

The first digestive movements in the embryo are mediated by mechanosensitive smooth muscle calcium waves

Peristalsis enables transport of the food bolus in the gut. Here, I show by dynamic ex vivo intra-cellular calcium imaging on living embryonic gut explants that the most primitive form of peristalsis that occurs in the embryo is the result of inter-cellular, gap-junction-dependent calcium waves that propagate in the circular smooth muscle layer. I show that the embryonic gut is an intrinsically mechanosensitive organ, as the slightest externally applied mechanical stimulus triggers contractile waves. This dynamic response is an embryonic precursor of the ‘law of the intestine’ (peristaltic reflex). I show how characteristic features of early peristalsis such as counter-propagating wave annihilation, mechanosensitivity and nucleation after wounding all result from known properties of calcium waves. I finally demonstrate that inter-cellular mechanical tension does not play a role in the propagation mechanism of gut contractile waves, unlike what has been recently shown for the embryonic heartbeat. Calcium waves are a ubiquitous dynamic signalling mechanism in biology: here I show that they are the foundation of digestive movements in the developing embryo. This article is part of the Theo Murphy meeting issue on ‘Mechanics of development’.