Longitudinal Muscle Layer Research Articles

CLSM Analysis of the Phalloidin-Stained Muscle System of the Nemertean Proboscis and Rhynchocoel

The proboscis and rhynchocoel musculature of 56 nemertean species was studied using phalloidin labelling and confocal laser scanning microscopy. Six types of muscle layers are found in the anterior proboscis of the nemerteans: inner circular, inner diagonal, inner longitudinal, outer diagonal, outer circular, and outer longitudinal. Only the inner circular and inner longitudinal muscle layers are present in all the nemerteans studied. Ten types of arrangement of the proboscis musculature are described. Three primary types ('palaeotype', 'heterotype', and 'hoplotype') correspond to the three nemertean supergroups (Palaeonemertea, Heteronemertea, and Hoplonemertea). The evolutionary transformations of the initial 'palaeotype' proboscis proceeded in two primary ways: increasing bilateral symmetry (Callinera, Hubrechtella, and most of Heteronemertea) and increasing polyradial symmetry (Baseodiscidae, Oxypolellinae, and Hoplonemertea). The musculature of the middle portion of the proboscis differs among the three groups with armature: Palaeonemertea (genus Callinera), Polystilifera, and Monostilifera. The musculature of the stylet apparatus of the monostiliferous nemerteans is more complicated than that of the polystiliferous nemerteans, and consists of four muscle components--basal and anterior sphincters, radial and longitudinal musculature. Among the studied monostiliferans, the different components of the stylet musculature are developed to varying degrees. In addition, data on the structure of the rhynchocoel with interwoven musculature are provided. The taxonomic significance and phylogenetic interpretation of the proboscis and rhynchocoel musculature is discussed.

Differences in time to peak carbachol-induced contractions between circular and longitudinal smooth muscles of mouse ileum.

The muscular layer in the GI tract consists of an inner circular muscular layer and an outer longitudinal muscular layer. Acetylcholine (ACh) is the representative neurotransmitter that causes contractions in the gastrointestinal tracts of most animal species. There are many reports of muscarinic receptor-mediated contraction of longitudinal muscles, but few studies discuss circular muscles. The present study detailed the contractile response in the circular smooth muscles of the mouse ileum. We used small muscle strips (0.2 mm × 1 mm) and large muscle strips (4 × 4 mm) isolated from the circular and longitudinal muscle layers of the mouse ileum to compare contraction responses in circular and longitudinal smooth muscles. The time to peak contractile responses to carbamylcholine (CCh) were later in the small muscle strips (0.2 × 1 mm) of circular muscle (5.7 min) than longitudinal muscles (0.4 min). The time to peak contractile responses to CCh in the large muscle strips (4 × 4 mm) were also later in the circular muscle (3.1 min) than the longitudinal muscle (1.4 min). Furthermore, a muscarinic M2 receptor antagonist and gap junction inhibitor significantly delayed the time to peak contraction of the large muscle strips (4 × 4 mm) from the circular muscular layer. Our findings indicate that muscarinic M2 receptors in the circular muscular layer of mouse ileum exert a previously undocumented function in gut motility via the regulation of gap junctions.

Cellular mediators of nitrergic neurotransmission in GI smooth muscles: no easy answer.

Gastrointestinal muscles receive both excitatory and inhibitory innervation, and both positive and negative influences impart important regulatory control of motility and transit. Twenty‐five years after nitric oxide (NO) was identified as a key neurotransmitter of enteric inhibitory motor neurons (Bult et al. 1990), the cells mediating nitrergic motor effects and the cellular mechanisms responsible for relaxation remain controversial. Three cell types found within GI muscles, i.e. smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and PDGFRα+ cells, form an electrical syncytium (the SIP syncytium) that integrates intrinsic and regulatory inputs (Sanders et al. 2014). A recent paper in The Journal of Physiology by Lies and coworkers addresses the question of which cell types mediate nitrergic motor responses in the longitudinal muscle layer of the murine colon (Lies et al. 2015). The authors used cell‐specific, tamoxifen‐inducible expression of Cre recombinase (iCre) in SMCs or ICC bred to mice with floxed Gucy1b3 (Gucy1b3flox/flox) to control expression of β1 subunits of soluble guanylate cyclase (GC), an essential component of NO receptors. The rationale was that elimination of functional receptors in ICC or SMCs would reveal the relative importance of these cells in nitrergic responses.

Anatomical, histological and histomorphometric study of the intestine of the northern pike (Esox lucius).

Background: The northern pike Esox lucius is a fresh water species belonging to the Esocidae family. It is a carnivorous fish which mostly feeds on invertebrates and fishes. The morphology of its intestine is very useful for understanding the fish’s digestive physiology, diagnosing some intestinal diseases and formulating suitable feeds. OBJECTIVES: This study was designed to determine the anatomical, histological and histomorphometric properties of the intestine of E. lucius. METHODS: The intestines of five E. lucius were examined in this study. After anatomical dissection, the histological specimens were taken and fixed in 10% formalin. Then, tissue passages were stained with hematoxylin-eosin, and Masson’s trichrome. RESULTS: The anatomical examination showed the short intestine with intestinal coefficient 0.68±0.09 in E. lucius which is a characteristic of the carnivorous species. The histological study revealed that the intestinal wall of E. lucius is composed of tunica mucosa, submucosa, muscularis, and serosa. The muscularis mucosa was not visible in the intestine. The stratum compactum is present between tunica mucosa and tunica submucosa. The histomorphometric results differentiated between three parts in the intestine of E. lucius namely anterior, middle and posterior. The maximum height of mucosal folds was observed in the anterior intestine due to its role in nutrient absorption. The mucosal fold’s height then decreased towards the posterior intestine. The tunica muscularis is significantly thicker in the anterior intestine, and the circular muscle layer is thicker than the longitudinal muscle layer throughout the entire length of the intestine. The posterior intestine possessed large numbers of goblet cells in comparison with other parts of the intestine, to promote elimination of unabsorbed particles. CONCLUSIONS: The results of this study revealed adaptation for the species feeding habits, so as to protect the intestine and increase absorptive processes.

Histochemical Study of Heparin-positive Mast Cells in the Terminal Part of Porcine Ductus Choledochus and Papilla Duodeni Major.

The study presented in detail the localization and density of mast cells (MCs) in the intramural part of the common bile duct (CBD) and in the major duodenal papilla (MDP) of domestic swine. MCs' density (number/mm(2) ) in different layers of both of the duct and papilla was evaluated after toluidine blue staining. Their number was higher in the lamina propria mucosae than in the tunica muscularis of the studied structures. The localization of berberine-positive, (heparin containing) MCs and the ratio between them and toluidine blue-positive MCs with γ-ma metachromasia was also established. Ratios of heparin-containing MCs in comparison with all toluidine blue-positive MCs were found as follows: ductus choledochus - 32% in the subglandular connective tissue of lamina propria mucosae in the intramural part of the duct; m. sphincter ductus choledochus - 31% in the circular and 0.06% in the longitudinal muscle layer; subserosa - 59%; papilla duodeni major - 0.03% in the subepithelial connective tissue and 34% in the subglandular connective tissue of lamina propria mucosae, respectively. The established large difference in heparin-positive MCs in both the subepithelial and subglandular connective tissues of CBD and MDP, respectively, is an evidence for the existence of mucosal and connective tissue MCs.

Stimulation of synthesis and release of brain-derived neurotropic factor from intestinal smooth muscle cells by substance P and pituitary adenylate cyclase-activating peptide.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. The expression and secretion of BDNF from smooth muscle cultured from the rabbit intestinal longitudinal muscle layer in response to substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was measured by western blot and enzyme-linked immunosorbent assay. BDNF mRNA was measured by reverse-transcription polymerase chain reaction. The expression of BNDF protein and mRNA was greater in smooth muscle cells (SMCs) from the longitudinal muscle than from circular muscle layer. PACAP and SP increased the expression of BDNF protein and mRNA in cultured longitudinal SMCs. PACAP and SP also stimulated the secretion of BDNF from cultured longitudinal SMCs. Chelation of intracellular calcium with BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prevented SP-induced increase in BDNF mRNA and protein expression and SP-induced secretion of BDNF. Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in SMCs and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease.

Circular and longitudinal muscles shortening indicates sliding patterns during peristalsis and transient lower esophageal sphincter relaxation.

Esophageal axial shortening is caused by longitudinal muscle (LM) contraction, but circular muscle (CM) may also contribute to axial shortening because of its spiral morphology. The goal of our study was to show patterns of contraction of CM and LM layers during peristalsis and transient lower esophageal sphincter (LES) relaxation (TLESR). In rats, esophageal and LES morphology was assessed by histology and immunohistochemistry, and function with the use of piezo-electric crystals and manometry. Electrical stimulation of the vagus nerve was used to induce esophageal contractions. In 18 healthy subjects, manometry and high frequency intraluminal ultrasound imaging during swallow-induced esophageal contractions and TLESR were evaluated. CM and LM thicknesses were measured (40 swallows and 30 TLESRs) as markers of axial shortening, before and at peak contraction, as well as during TLESRs. Animal studies revealed muscular connections between the LM and CM layers of the LES but not in the esophagus. During vagal stimulated esophageal contraction there was relative movement between the LM and CM. Human studies show that LM-to-CM (LM/CM) thickness ratio at baseline was 1. At the peak of swallow-induced contraction LM/CM ratio decreased significantly (<1), whereas the reverse was the case during TLESR (>2). The pattern of contraction of CM and LM suggests sliding of the two muscles. Furthermore, the sliding patterns are in the opposite direction during peristalsis and TLESR.

A simple, inexpensive model to demonstrate how contraction of GI longitudinal smooth muscle promotes propulsion.

peristalsis is a propulsive activity that involves both circular and longitudinal muscle layers of the esophagus, distal stomach, and small and large intestines. During peristalsis, the circular smooth muscle contracts behind (on the orad side) the bolus and relaxes in front (on the aborad side) of

New Insights into the Genetics of Fetal Megacystis: ACTG2 Mutations, Encoding γ-2 Smooth Muscle Actin in Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (Berdon Syndrome)

Objective: To identify the molecular basis for prenatally suspected cases of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) (MIM 249210) in 3 independent families with clinical and radiographic evidence of MMIHS. Methods: Whole-exome sequencing (WES) and Sanger sequencing of the ACTG2 gene. Results: We identified a novel heterozygous de novo missense variant in ACTG2 c.770G>A (p.Arg257His) encoding γ-2 smooth muscle actin (ACTG2) in 2 siblings with MMIHS, suggesting gonadal mosaicism of one of the parents. Two additional de novo missense variants (p.Arg257Cys and p.Arg178His) in ACTG2 were identified in 2 additional MMHIS patients. All of our patients had evidence of fetal megacystis and a normal or slightly increased amniotic fluid volume. Additional findings included bilateral renal hydronephrosis, an enlarged fetal stomach, and transient dilated bowel loops. ACTG2 immunostaining of the intestinal tissue showed an altered muscularis propria, a markedly thinned longitudinal muscle layer, and a reduced amount and abnormal distribution of ACTG2. Conclusion: Our study demonstrates that de novo mutations in ACTG2 are a cause of fetal megacystis in MMIHS and that gonadal mosaicism may be present in a subset of cases. These findings have implications for the counseling of families with a diagnosis of fetal megacystis with a preserved amniotic fluid volume and associated gastrointestinal findings.

The making of an octopus arm.

BackgroundMost of our current findings on appendage formation and patterning stem from studies on chordate and ecdysozoan model organisms. However, in order to fully understand the evolution of animal appendages, it is essential to include information on appendage development from lophotrochozoan representatives. Here, we examined the basic dynamics of the Octopus vulgaris arm’s formation and differentiation - as a highly evolved member of the lophotrochozoan super phylum - with a special focus on the formation of the arm’s musculature.ResultsThe octopus arm forms during distinct phases, including an early outgrowth from an epithelial thickening, an elongation, and a late differentiation into mature tissue types. During early arm outgrowth, uniform proliferation leads to the formation of a rounded bulge, which subsequently elongates along its proximal-distal axis by means of actin-mediated epithelial cell changes. Further differentiation of all tissue layers is initiated but end-differentiation is postponed to post-hatching stages. Interestingly, muscle differentiation shows temporal differences in the formation of distinct muscle layers. Particularly, first myocytes appear in the area of the future transverse prior to the longitudinal muscle layer, even though the latter represents the more dominant muscle type at hatching stage. Sucker rudiments appear as small epithelial outgrowths with a mesodermal and ectodermal component on the oral part of the arm. During late differentiation stages, cell proliferation becomes localized to a distal arm region termed the growth zone of the arm.ConclusionsO. vulgaris arm formation shows both, similarities to known model species as well as species-specific patterns of arm formation. Similarities include early uniform cell proliferation and actin-mediated cell dynamics, which lead to an elongation along the proximal-distal axis. Furthermore, the switch to an adult-like progressive distal growth mode during late differentiation stages is reminiscent of the vertebrate progress zone. However, tissue differentiation shows a species-specific delay, which is correlated to a paralarval pelagic phase after hatching and concomitant emerging behavioral modifications. By understanding the general dynamics of octopus arm formation, we established a basis for further studies on appendage patterning, growth, and differentiation in a representative of the lophotrochozoan super phylum.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-015-0012-8) contains supplementary material, which is available to authorized users.

HSPB8 and the Cochaperone BAG3 Are Highly Expressed During the Synthetic Phase of Rat Myometrium Programming During Pregnancy1

The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.

Sa1112 Three-Dimensional Myoarchitecture of Porcine Gastro-Esophageal Junction With Diffusion Tensor Imaging

Introduction: The anatomy and function of the gastro-esophageal junction (GEJ) is complex with mucosa-submucosa layers and smooth muscle layers organized into circular and longitudinal muscle layers. These layers continue from the esophagus into the GEJ and the stomach. Furthermore, the crura of the diaphragm and the lower esophageal sphincter are components of the GEJ. Little is known about the microstructure of the GEJ. Diffusion tensor imaging (DTI) allows mapping of the diffusion process of molecules, mainly water, in biological tissues. Water molecule diffusion patterns can therefore reveal microscopic details about tissue architecture. Its main clinical application has been in the study of neurological disorders such as acute stroke. Until now DTI has not be used much in esophagology. Aims: This study aims to reconstruct an anatomically realistic 3D model of the GEJ on the basis of DTI and more specifically to distinguish the muscle fiber transition between the esophagus, the stomach, and the diaphragmatic crura. Methods: Three 40-60kg pigs were used for obtaining the GEJ and adjacent parts of the esophagus, stomach and diaphragm. The pig was sacrificed during deep anesthesia before opening the abdominal and thoracic cavities. The intestines were removed whereas the liver and stomach was kept in place for maintaining the anatomical position of GEJ. The abdominal cavity and thoracic cavities were filled with formalin and closed. After 24h of tissue fixation, the pigs were reopened and the esophagus, upper part of stomach and intact diaphragm were dissected out and fixed with 4% formalin in an organ bath for further 2-3 days. A 7x7x7cm3 block including the entire GEJ region and lower part of the esophagus, upper part of the stomach, and the diaphragmatic crura was prepared for Magnetic Resonance (MR) scanning. Scanning was done in a Philips Intera, 1.5T MR scanner for obtaining anatomy imaging and DTI. The wall thickness at different layers of the GEJ as well as the helical angles of the longitudinal muscle fiber, the circular muscle fiber, the gastric sling fiber, gastric oblique fibers and diaphragm crura fiber along the central axis of the GEJ were calculated on the basis of the anatomic imaging and DTI. Results: It was possible to visualize the anatomical features of GEJ using DTI. The figure shows the total muscle fibers 3D distribution along the GEJ in a traced DT image. Differences in diffusion properties between the GEJ structures such as the sling fibers were easy to recognize and detailed layer data were obtained and used as input for an ongoing development of a realistic functional model of the GEJ. Conclusions and perspectives: This study clearly demonstrated that DTI may be useful in esophagology and moved a step further by establishing an anatomical-functional realistic computer model of the GEJ.

Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.

Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-MΦ/DCs corrected the altered neuronal phenotype of TLR2(-/-) mice. Supplementation of TLR2(-/-) neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2(-/-) neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategies.

Forkhead Box F2 Regulation of Platelet-derived Growth Factor and Myocardin/Serum Response Factor Signaling Is Essential for Intestinal Development

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2(-/-) mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP(+/-));Foxf2(-/-) mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling.

Periodic oscillatory myoelectrical activity in the gut

Introduction: Myoelectrical (ME) activity, including slow waves and spikes, modulate a variety of patterns of intestinal motility. Any abnormality in the generation and propagation of migrating ME complexes leads to uncoordinated contractions of smooth muscle syncytia, responsible for a clinical condition classified as intestinal dysrhythmia. Objectives: To investigate the dynamics of myoelectrical patterns in the wall of the small bowel under normal and pathological conditions. Materials and methods: The study applied a computer model to analyse electromechanical wave phenomena in a functional unit of the gut. The computer model was based on anatomical and physiological data obtained from in vivo and in vitro experiments. During the numerical experiments the spatial distribution and the dynamics of ME patterns, frequencies, velocities and active forces were recorded. Results: Results of numerical experiments revealed periodic oscillatory activity in the longitudinal smooth muscle layer of the organ. This depended on pacemaker settings and intrinsic electrical properties of muscle syncytia. The observed self-sustained ME activity might underlie the pathogenesis of intestinal dysrhythmia. Conclusions: A detailed explanation for the observed event cannot be provided at present. We suggest that the abnormal oscillatory activity of interstitial cells of Cajal and histomorphological arrangements among muscle cells contribute to the outcome. Acknowledgements: Arabian Gulf University for sponsoring the research.

Neurochemical features of endomorphin-2-containing neurons in the submucosal plexus of the rat colon.

To investigate the distribution and neurochemical phenotype of endomorphin-2 (EM-2)-containing neurons in the submucosal plexus of the rat colon. The mid-colons between the right and left flexures were removed from rats, and transferred into Kreb's solution. For whole-mount preparations, the mucosal, outer longitudinal muscle and inner circular muscle layers of the tissues were separated from the submucosal layer attached to the submucosal plexus. The whole-mount preparations from each rat mid-colon were mounted onto seven gelatin-coated glass slides, and processed for immunofluorescence histochemical double-staining of EM-2 with calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), nitric oxide synthetase (NOS), neuron-specific enolase (NSE), substance P (SP) and vasoactive intestinal peptide (VIP). After staining, all the fluorescence-labeled sections were observed with a confocal laser scanning microscope. To estimate the extent of the co-localization of EM-2 with CGRP, ChAT, NOS, NSE, SP and VIP, ganglia, which have a clear boundary and neuronal cell outline, were randomly selected from each specimen for this analysis. In the submucosal plexus of the mid-colon, many EM-2-immunoreactive (IR) and NSE-IR neuronal cell bodies were found in the submucosal plexus of the rat mid-colon. Approximately 6 ± 4.2 EM-2-IR neurons aggregated within each ganglion and a few EM-2-IR neurons were also found outside the ganglia. The EM-2-IR neurons were also immunopositive for ChAT, SP, VIP or NOS. EM-2-IR nerve fibers coursed near ChAT-IR neurons, and some of these fibers were even distributed around ChAT-IR neuronal cell bodies. Some EM-2-IR neuronal cell bodies were surrounded by SP-IR nerve fibers, but many long processes connecting adjacent ganglia were negative for EM-2 immunostaining. Long VIP-IR processes with many branches coursed through the ganglia and surrounded the EM-2-IR neurons. The percentages of the EM-2-IR neurons that were also positive for ChAT, SP, VIP or NOS were approximately 91% ± 2.6%, 36% ± 2.4%, 44% ± 2.5% and 44% ± 4.7%, respectively, but EM-2 did not co-localize with CGRP. EM-2-IR neurons are present in the submucosal plexus of the rat colon and express distinct neurochemical markers.

Embryonic Diffrentiation of the Colon of One Humped Camel (Camelus Dromedarius): A Histomorphology

A study involving histomorphological differentiation was conducted on the colon of 35 foetuses (both sex) of the one-humped camel collected from the Sokoto metropolitan abattoir, over a period of five months at different gestational ages. The approximate age of the fetuses was estimated and categorized into first, second and third trimester. Grossly, the color of the large intestine was whitish at first trimester and grayish to white in second and third trimester. The colon was divided into three main portions namely the ascending colon, the coiled and the descending part which formed the long part of the colon in second and third trimesters. At first trimester the coiled part was not differentiated but divided into two parts such as the centripetal and centrifugal part at second and third trimester. Histological observation of the tissues in this study revealed a complete structure of the tubular organ. The colon was found to consist of four layers namely: Tunica mucosa, Tunica sub mucosa, Tunica muscularis and Tunica serosa. The epithelium of the Tunica mucosa was stratified squamous epithelium with varying degree of stratification at first trimester and transformed to low columnar /cuboidal epithelium at second trimester. At third trimester, the epithelium was simple columnar epithelium. The lamina propria mucosa was found absent at first trimester but prominent at second and third trimester. The Lamina muscularis mucosa was found prominent at third trimester but not identified at first and second trimester. At first trimester of age tunica submucosa was prominent while at second trimester, it consisted of connective tissue cells and fibres scattered all over the layers with preliminary blood vessels. The cells and fibres were undifferentiated at this stage. There was no evidence of lymphatic nodular cells within the layer. At third trimester of age, the connective tissues and blood vessels were found prominent and the lymphatic nodular cells were found throughout the length of the colon. The tunica muscularis of camel colon consist of inner skeletal and outer longitudinal smooth muscle layers. At first trimester this layer did not differentiate into these two zones but only longitudinal orientation of smooth muscle layer. At second trimester, the layers of two zones with clear demarcation were observed. A thin layer of connective tissue comprising of undifferentiated cells lined the colon externally was observed in all the stages of development. Based on the above findings, it showed that development of the camels’ colon was histologically in succession and different from other domestic animals by having an extensive skeletal muscle at the tunica muscularis.

Minimally invasive hybrid surgery combined with endoscopic and thoracoscopic approaches for submucosal tumor originating from thoracic esophagus.

BackgroundDespite the efficacy of molecular targeted therapy, surgical resection remains the only curative primary treatment for gastrointestinal stromal tumors (GISTs). However, in cases when the tumor originates from the thoracic esophagus, conventional transthoracic approach is highly invasive.MethodsAll procedures were performed with patients in a prone position through a double-lumen endotracheal tube for single-lung ventilation. First, to clarify the resection layer between the tumor and mucosal layer of the esophagus, a sodium hyaluronate solution colored with indigo carmine was injected into the submucosa via the esophagoscopic approach. Second, we thoracoscopically divided the longitudinal muscle of the esophagus and enucleated the tumor through three ports by dissecting along the artificially colored submucosa, thereby minimizing accidentally opening of the esophageal mucosa. Third, we sutured the divided longitudinal muscle layer and removed the tumor from the thoracic cavity.ResultsFour tumors, including one GIST, were successfully resected via this hybrid approach. The mean surgical time was 137.7 min (range, 60–231 min), and the mean blood loss was 21.2 ml (range, 3–65 ml). No perioperative complications occurred, including with accidental opening of the esophageal mucosa.ConclusionsOur minimally invasive hybrid surgery combined with esophagoscopic and thoracoscopic approaches demonstrated successful resection. This surgery could have advantages both for curing esophageal submucosal tumor and for minimizing surgical invasiveness.

Immunohistochemistry of Heat Shock Protein 70 (Hsp70) on Earthworm (Lumbricus terrestris) in Gold Mining Sites

The traditional gold mining in Cempaka, Banjarbaru, South Kalimantan, Indonesia used mercury in order to separate the gold with a fine sand and rocks. Mercury can impair the function of the cellular and organism physiology that live in the mining sites. In cellular on earthworm may adapt to help in repairing cell damage with expression of HSP70 proteins. This study was conducted in November 2012 to September 2014. Sampling of soil and earthworms do in Banjarbaru village (control) and in paddy field near gold mining among them Murung village, Pumpung village and Palam village (ex. PT. Galuh Cempaka). Soil quality in the gold mining indicate conditions are still good in term of C-organic, pH and CEC. The C-organic content of the soil between the 2.08-2.52%; the value of pH H 2 O between 4.90-5.72, CEC between 17.20-18.11. Detection of protein HSP70 on earthworms use the method immunohistochemistry. HSP70 expression was found in the longitudinal muscle layer (LM). The expression of HSP70 most occurred in earthworms that live in the land of the paddy fields ex. PT. Galuh Cempaka.

Skin-derived precursors generate enteric-type neurons in aganglionic jejunum

PurposeSkin-derived precursor cells (SKPs) may regenerate the enteric nervous system in Hirschsprung’s disease. SKPs migrate and differentiate into myenteric ganglia in aganglionic intestine. We sought to characterize the time-course of SKP gangliogenesis and enteric neurotransmitter synthesis in vivo. MethodsAdult Lewis rat jejunal segments were isolated and denervated with benzalkonium chloride (BAC). Denervation was evaluated by immunohistochemical (IHC) stains for markers of mature neuronal and glial cells. Green fluorescent protein (GFP)-expressing neonatal rat SKPs were cultured in neuroglial-selective medium. SKPs were transplanted into aganglionic segments 65–85days after BAC treatment. IHC was performed to identify glia, neurons, and neurotransmitter synthesis in GFP+cells between post-transplant days 1 and 28. ResultsAganglionosis was confirmed by IHC. On post-transplant days 1 and 2, GFP+cells were detected near injection sites within the muscularis propria. GFP+cell clusters were evident only between longitudinal and circular smooth muscle layers at post-transplant days 14, 21, and 28. These structures co-expressed markers of mature neurons and gliocytes. Several markers of neurotransmitter synthesis were detected in GFP+clusters at days 21 and 28. ConclusionSKPs are capable of enteric neuroglial differentiation in vivo. SKPs migrate to the intermuscular layer of aganglionic intestine within days of transplantation. Our observations suggest that SKPs are capable of generating enteric ganglia in aganglionic intestine.