Rat Esophagus Research Articles

МОРФОФУНКЦІОНАЛЬНІ ХАРАКТЕРИСТИКИ СТРАВОХОДУ ЗА УМОВ ЛУЖНОГО ОПІКУ СТРАВОХОДУ 2 СТУПЕНЯ ТА ПРИ ВВЕДЕННІ ЕКСТРАКТУ СТРУЧКІВ PHASEOLUS VULGARIS

The most widespread chemical damage in children – burn of the esophagus. It occurs when accidentally swallowing substances that have in their composition alkali, acid or other caustic substances. The depth of burn depends on the concentration and time of contact of the mucous membrane with the affecting agent. As a result of penetration of alkalis and contact with the wall of the esophagus, along with necrosis, damage to the mucous membrane, submucosal and muscular membrane of the esophagus occurs. Despite a large number of drugs that have been tested in the treatment of complications caused by esophageal burns, there is little information that would indicate the high effectiveness of the previously used drugs. The analysis of literature data shows the positive influence of polyphenols of natural origin on the normalization of physiological and biochemical parameters in various pathologies. The extract of common beans (Phaseolus vulgaris pods extract (PVPE)) is an example of a rich bioactive component of the source with beneficial characteristics for humans. Therefore, the purpose of our work was to investigate the effect of the Phaseolus vulgaris pods extract on the healing of post-burns wounds. When morpho-functional characteristics of the esophagus changes in the conditions of alkaline burn of the esophagus of the 2nd degree, it was shown that at 1 day of the experiment there is a violation of the mucous membrane, which manifests itself in the destruction of the multilayered epithelium, namely, in the submucous membrane there is a significant accumulation of leukocytes, leukocytic infiltration is fixed in the partly destroyed muscle layer. At 31 days of study in a group of animals with burn of the esophagus there is a certain restoration of the integrity of the mucous membrane of the esophagus, mainly with hyperplasia; there is also a decrease in the presence of leukocytes; however, there is an enlargement of the connective tissue in the submucosal membrane, which may indicate the formation of a scar. The data of the morphological description are confirmed by the morphometric parameters of the calculation of the stenosis index. With the use of extracts of beans under chemical burns esophagus conditions, improvement of the morpho-functional status of the esophagus of rats was noted: the formed connective tissue scar does not significantly affect the stenosis index (its significance is the intermediate position); the high level of leukocyte infiltration is reduced to the control parameters, which may indicate the obvious therapeutic effect of the extract of beans pods at alkaline burns of the esophagus.

Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Results: The histological analysis revealed that only the first decellularization cycle was enough to produce a cellular and nuclei free esophageal scaffold with a proper extracellular matrix orientation. These results were further confirmed by biochemical quantification. Conclusions: Based on the above results, the current decellularization protocol can be applied successfully in order to produce an esophageal tissue engineered construct.

Na+ /Ca2+ exchanger 1 is a key mechanosensitive molecule of the esophageal myenteric neurons.

Our earlier studies showed that mechanical stretch activates inhibitory motor neurons of the oesophagus; however, the underlying molecular mechanisms are unclear. Here, we sought to examine if Na+ /Ca2+ exchanger 1 (NCX1) is responsible for the mechanosensitivity in the esophageal myenteric neurons (EMN) of rats and humans. The function of NCX1 in primary culture of neurons was determined using calcium imaging, and mechanosensitivity was tested using osmotic stretch and direct mechanical stretch. Axial stretch-induced relaxation of the lower esophageal sphincter (LES) was also studied in vivo in rats. The expression and co-localization of NCX1 with nNOS were identified in the EMN from both rats and humans. The extracellular Ca2+ entry caused by ATP through purinergic signalling in the rat EMN was significantly inhibited by selective NCX blockers. Removal of extracellular Na+ to activate the Ca2+ entry mode of NCX1 induced an increase in the cytoplasmic calcium ([Ca2+ ]cyt ), which was attenuated by NCX blockers. Osmotic stretch and mechanical stretch-induced [Ca2+ ]cyt signalling in the rat and human EMN were attenuated by NCX blockers as well as specific NCX1 knockdown. Osmotic stretch and mechanical stretch also induced [Ca2+ ]cyt signalling in the Chinese hamster ovary (CHO) cells with NCX1 over-expression, which was attenuated by NCX blockers. Finally, NCX blockade inhibited axial stretch-activated LES relaxation in vivo experiments in the rats. We demonstrate a novel NCX1/Ca2+ pathway in the mechanosensitive neurons of rat and human oesophagus, which may provide a potential therapeutic target for the treatment of oesophageal motility disorders.

A nutrigenetic approach for investigating the chemopreventive effects of black raspberries during the development of preneoplastic esophagi in rats.

Large epidemiological studies have shown that diets high in fruits reduce the risk of esophageal squamous cell carcinoma (ESCC). The current study investigated the effects of black raspberries (BRBs) on gene expression during the development of preneoplastic esophagi in rats. Using a post-initiation protocol, F344 rats were injected with N-nitrosomethylbenzylamine (NMBA) and then fed either a control diet or 5% BRBs. At weeks 9, 15, and 35, we euthanized subgroups of the rats and collected preneoplastic esophagi to isolate RNA samples for DNA microarray. Along the development of NMBA-induced preneoplastic esophagi, NMBA injections led to differential expression of 1181 genes comparing to control rats, and dietary BRBs modulated 428 genes in esophagi from NMBA-treated rats. There are 137 common genes between 1181 and 428 gene sets, and BRBs significantly reversed the expression of 133 genes. These genes are associated with multiple gene oncology functions. BRBs induced an 8.8-fold gene enrichment on the pathway of inflammatory response and regulated 10 genes involved in this pathway. Among them, BRBs significantly reversed the expression of pro-inflammatory cytokines, such as CCL2, S100A8, and IL19. BRBs exhibit strong anti-inflammatory effects against NMBA-induced rat esophageal tumorigenesis.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage

African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56patients and analyzed DNA samples from patients to identifyassociated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Impairment of rat oesophageal muscle contractility associated with experimental non-erosive oesophageal mucosal damage.

What is the central question of this study? Is the responsiveness of isolated segments of the rat oesophagus to contractile or relaxant stimuli susceptible to acute luminal exposure of the oesophagus to an acid solution that contains pepsin and bile salt? What is the main finding and its importance? The study reveals that luminal acidity is an important factor that disrupts barrier function in the oesophagus to allow the diffusion of noxious agents, such as bile acid, that alter the contractile status of the oesophageal body, even in the absence of inflammation. We investigated whether the experimental simulation of duodenogastro-oesophageal reflux alters the contractile responsiveness of rat oesophageal strips. After 30min of luminal exposure to a solution at acid pH that contained pepsin and taurodeoxycholic acid, isolated strips of the rat oesophagus and gastro-oesophageal junction were subjected to contractile or relaxing stimuli. Acid challenge decreased the responsiveness of oesophageal strips to contractile stimulation, especially in oesophageal preparations that were mounted following the circular orientation of the muscularis externa layer. The contractility of longitudinal preparations of the rat oesophagus appeared less susceptible to the deleterious effects of acid challenge. In contrast, the responsiveness of ring-like preparations from the gastro-oesophageal junction to contractile stimulation was unaltered by acid challenge. Taurodeoxycholic acid decreased the responsiveness of circular oesophageal preparations to KCl, an effect that was exacerbated by luminal acidity. On the contrary, although the relaxant ability of the rat oesophagus did not change, acid challenge increased the relaxant efficacy of sodium nitroprusside and isoprenaline in strips of the gastro-oesophageal junction. A significant decrease in transepithelial electrical resistance was seen when the oesophageal mucosa was challenged at pH1 but not at pH4. Treatment with alginate blunted the deleterious effects of acid challenge on transepithelial electrical resistance and the responsiveness of oesophageal preparations to KCl. The present findings support the notion that luminal acidity is an important factor that disrupts barrier function in the oesophagus to allow the diffusion of noxious agents, such as bile acid, that alter the contractile status of the oesophagus.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Protective antioxidant effect of melanin against chemical burn-induced esophageal injury

Background: Oxidative stress is the main cause of mortality in chemical burn-induced esophageal injury. Melanin, a natural antioxidant compound from yeast-like fungus Nadsoniella nigra strain X- 1, has been shown to decrease the content of lipid peroxidation products after burn. The aim of this study was to investigate the protective effect of melanin in the treatment of esophageal injury after a chemical burn.
 Methods: A alkali burn model was used to induce injury to the esophagus in immature rats. Changes in the levels of malondialdehyde, secondary products of lipid peroxidation (thiobarbituric acid reactive substances), superoxidase dismutase, and catalase in the blood, as well as changes in the esophagus tissue, were examined.
 Results: Melanin decreased the content of lipid peroxidation products following burn injury. Melanin increased the activity of superoxidase dismutase and reduced the activity of catalase, as well as reduced esophagus oxidative injury in our chemical burn model.
 Conclusion: Melanin treatment may protect against chemical burninduced esophageal injury, possibly by inhibiting burn-induced oxidative stress.

Acidic Bile Salts Induce Epithelial to Mesenchymal Transition via VEGF Signaling in Non-Neoplastic Barrett’s Cells

Metaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues. We compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE. We detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues. In BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia.

An improved surgical procedure to establish a gastroesophageal reflux model with a high incidence of Barrett's esophagus in rats.

Barrett's esophagus (BE) is a complication of gastroesophageal reflux disease and is a precursor lesion of esophageal adenocarcinoma. In existing BE models, the incidence of BE is typically low and induction is usually time consuming. In the present study, a gastroesophageal reflux model with a high incidence of BE in rats. Rats were divided into a model group and a sham operation group, and anesthetized with an inhalation anesthesia machine. Stomach-jejunal anastomosis (SJA) and esophagus-jejunal anastomosis (EJA) were simultaneously performed in the model group. The distance between the Treitz ligament and the gastro-jejunal anastomosis was shortened to 3 cm. The distance between the SJA and the EJA was prolonged to 1–1.5 cm. However, 15/40 rats in the model group succumbed to post-surgical complications (mortality rate was 37.5%). The weight of surviving rats in the model group was significantly lower compared with the sham group rats post-surgery. Erosions and ulcers were common of the surviving rats in the model group, with an incidence of 80% (20/25). Squamous cell dysplasia was identified in 40% (10/25) of rats in model group. The modified model was well established within 16 weeks. Notably, the modified surgical procedure used enhanced the incidence of BE in rats from 47% in the EJGJ model (as establish by Zhang) to 100%. To conclude, this model can be used as a reliable animal model for basic research on BE.

Nanofunctionalized Stent-Mediated Local Heat Treatment for the Suppression of Stent-Induced Tissue Hyperplasia.

Current therapeutic strategies are insufficient for suppressing stent-induced restenosis. Here, branched gold nanoparticles (BGNP)-coated self-expandable metallic stents (SEMSs) were developed for a local heat-induced suppression of stent-related tissue hyperplasia. Our polydopamine (PDA) coating on SEMS allowed BGNP crystal growth on the surface of SEMSs. The prepared BGNP-coated SEMS showed effective local heating under near-infrared laser irradiation. The effectiveness of BGNP-coated SEMSs for suppressing stent-related tissue hyperplasia was demonstrated in a rat esophageal model ( n = 52). BGNP-coated SEMS placement under fluoroscopic guidance was technically successful in all rats. The placed BGNP-coated SEMS in rat esophagus achieved three different local heat dose ranges (50, 65, and 80 °C) under fluoroscopic image-guided local irradiation. Follow-up endoscopic examination readily monitored the local heating and observed significantly decreased tissue hyperplasia at 4 weeks of local heat treatments (50 and 65 °C). Finally, Western blot, histology, immunohistochemistry (HSP70, αSMA, and TUNEL), and immunofluorescence (Ki67 and BrdU) analyses along with the statistical analysis confirmed that optimized BGNP-coated SEMS-mediated local heat treatments inducing the expression of anti-inflammatory HSP70 effectively suppresses tissue hyperplasia after stent placement in the esophagus. Our local heating with nanofunctionalized stents represents a promising new approach for suppressing stent-related tissue hyperplasia.

Caustic Lesions of Esophagus in Rats and Therapy with Stable Gastric Pentadecapeptide BPC 157

AimWe focused on caustic lesions of the esophagus in rat and therapy with the stable gastric pentadecapeptide BPC 157. Caustic lesions of the esophagus are one of the life‐threatening morbidities after ingestion of acid or alkali, as an accidental ingestion affecting mostly children. BPC 157 is an anti‐ulcer peptide, stable in human gastric juice, and shown as a novel mediator of Robert's cytoprotection with the beneficial effect in the whole gastrointestinal tract (Curr Pharm Des. 2017;23(27):4012–4028; Curr Pharm Des. 2014;20(7):1126–35; Curr Pharm Des. 2010;16(10):1224–34). Providing a direct cytoprotective effect of BPC 157 therapy on esophagus as well (Curr Pharm Des. 2010;16(10):1224–34), we assume its beneficial effect when given after caustic substance ingestion.MethodsDeeply anesthetized Albino Wistar rats, 200 g b.w., received directly 0.5 mL of the caustic substance (10% NaOH or 20% HCl respectively) at the proximal part of the esophagus with the free passage to the stomach, and immediately thereafter, BPC 157 10 μg/kg or saline (5 mL/kg) intraperitoneally. Assessment (macro/microscopy), was at the sacrifice, at 1, 2, 5 and 10 minutes. At 1 and 10 minutes, in proximal, middle and distal esophagus tissue samples, oxidative stress assessment was by thiobarbituric acid (TBA) reactivity as malondialdehyde equivalents (MDA). Likewise, at 1 and 10 minutes post‐injury, we determined nitric oxide (NO) levels in proximal, middle and distal esophagus tissue samples using the Griess reaction (Griess Reagent System, Promega, USA).ResultsGross assessment at all time intervals revealed a marked reduction of the lesions induced by both caustics in rats that received BPC 157 therapy. Microscopic examination was along with macroscopic evaluation. Thus, BPC 157‐rats exhibited markedly less damage in the proximal, middle and distal esophagus when challenged with caustics. Of note, in addition to the extensive esophageal caustic lesions, controls exhibited a huge increase of the oxidative stress and NO‐levels following caustic application. By contrast, rats damaged by caustic that subsequently received BPC 157 therapy had markedly less MDA and NO‐tissue level, even close to those values noted in the healthy tissue samples.ConclusionBPC 157 has a cytoprotective effect on the esophagus. Likewise, of particular importance is the evidence that this beneficial effect is obtained when the BPC 157 therapy is given after caustic ingestion. Thus, a practical importance of these findings should be emphasized.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effectiveness of Preimplantation Recellularization of Acellular Rat-Esophagus Matrices Using GFP-Positive Cells

The use of recellularized matrices allows obtaining tissue-engineered structures that largely reproduce the morphofunctional features of native organs and tissues. Doubts as to the advisability of preimplantation recellularization of the scaffold, which are associated with the death of prepopulation cells on the scaffold in vivo, require studying the behavior of cells on plastic and biological implants after recellularization. Rat esophagus that had undergone detergent-enzymatic decellularization was repopulated with GFP-positive cells intensely fluorescing in the green spectrum, which allowed us to trace the status of the initial cell population during cultivation on plastic and after transplantation, determine the viability and metabolic activity of cells, and conduct fluorescent detection of the cells on the scaffold before and after implantation. There were no data on the apparent cell proliferation on the matrix; however, there were indirect indications of metabolic activity and GFP synthesis by the cells that populated the scaffold before implantation.

An Ethanolic Extract of Allium hookeri Root Alleviates Reflux Esophagitis and Modulates NF-κB Signaling.

Reflux esophagitis (RE) is a kind of gastroesophageal reflux disease, of which an esophageal inflammatory lesion is caused by the contents of the stomach and duodenum flowing back into the esophagus. Allium hookeri is a plant possessing both nutritional and medicinal properties. In our study, we investigated the inhibition effect of inflammation of A. hookeri root extract (AHE) on inflammatory RAW264.7 macrophage cells induced by lipopolysaccharide and rat models of RE. The results showed that AHE significantly reduced the production of nitric oxide (NO) and the protein expression levels of various mediators related to inflammation including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Furthermore, AHE also inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) by inhibiting the phosphorylation IκBα. In addition, AHE administration significantly ameliorated esophageal mucosal damage upon histological evaluation of RE in rats. AHE was also found to downregulate the expression levels of proteins such as COX-2, TNF-α, and IL-1β in the rat esophagus. AHE markedly attenuated activation of NF-κB and phosphorylation of IκBα at the same time. These results indicated that AHE suppressed LPS-induced inflammatory responses in RAW264.7 cells and may help reduce the development of esophagitis through the modulation of inflammation by regulating NF-κB activation.

Matrix metalloproteinase functioning in case of esophagus acid burn

Despite the important role of Metalloproteinase (MMP) in β-cellular matrix remodeling there is clearly not enough data concerning the MMP proteolytic activity during first three weeks (early scar changes) of postoperative gastric ulcers. Thus the main aim of our work was the MMP, TIMP-1 and bFGF overall activity determination as well as their role in post-burn wound healing in case of the 2nd degree esophagus acid burn infliction. The greatest increase in the metalloproteinase activity and bFGF level was observed at 21st d of our experiment. In addition, the greatest increase in the TIMP-1 level in the esophageal mucosal was observed at 7th d after burn. These changes may be an evidence of chronic inflammatory processes and scar changes in rat esophagus after the EAB infliction.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

Super-achromatic monolithic microprobe for ultrahigh-resolution endoscopic optical coherence tomography at 800\u2009nm

Endoscopic optical coherence tomography (OCT) has emerged as a valuable tool for advancing our understanding of the histomorphology of various internal luminal organs and studying the pathogenesis of relevant diseases. To date, this technology affords limited resolving power for discerning subtle pathological changes associated with early diseases. In addition, it remains challenging to access small luminal organs or pass through narrow luminal sections without potentially causing trauma to tissue with a traditional OCT endoscope of a 1-1.5 mm diameter. Here we report an ultracompact (520 µm in outer diameter and 5 mm in rigid length) and super-achromatic microprobe made with a built-in monolithic fiber-optic ball lens, which achieves ultrahigh-resolution (1.7 µm axial resolution in tissue and 6 µm transverse resolution) for endoscopic OCT imaging at 800 nm. Its performance and translational potential are demonstrated by in vivo imaging of a mouse colon, a rat esophagus, and small airways in sheep.

Halofuginone improves caustic-induced oxidative injury of esophagus in rats.

The aim of this study is to evaluate the anti-inflammatory and anti-fibrotic effects of halofuginone in caustic esophageal burn injury in rats. Corrosive esophageal injury (CEI) was produced in male Wistar albino rats by instilling NaOH solution (1ml, 37.5%) into the distal esophagus. Rats were decapitated on the 3rd day (early group) or 28th day (late group), and treated daily with either saline or halofuginone (100µg/kg/day; i.p.), continued on alternate days after the third day. Histopathological evaluation and measurement of nitric oxide (NO), peroxynitrite (ONOO-) and oxygen-derived radicals by chemiluminescence (CL) were made in the distal 2cm of the esophagus. Non-irrigated proximal esophageal samples were assessed for the levels of nuclear factor (NF)-κB, caspase-3, glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) activity. GSH, MDA, NF-κB and caspase-3 levels, and MPO activity in the proximal esophagus were not different among groups. Increased number of TUNEL (+) cells in the irrigated esophagus of the early and late caustic injury groups was reduced by halofuginone treatment. High microscopic damage scores in both early and late CEI groups were decreased with halofuginone treatment. NO, ONOO- and CL levels, which were elevated in the saline-treated early CEI group, were reduced by halofuginone treatment, but reduced NO and ONOO- levels in the late period of saline-treated group were increased by halofuginone. In addition to its anti-fibrotic effects, current findings demonstrate that halofuginone exerts antioxidant and anti-apoptotic actions and supports therapeutic potential for halofuginone in CEI-induced oxidative stress.

Morphological evaluation of the quality of decellularized esophagus in rat

The main aim of our investigation was analysis and optimization of the existing protocols of esophagus decellularization in rats, identification of parameters affecting the quality of decellularization and intactness of the native properties of esophageal extracellular matrix (ECM). Materials and methods. We developed a modified decellularization protocol based on detergent-enzymatic method using sodium deoxycholate and DNAse. Results. Morphological evaluation of the obtained decellularized matrices demonstrated the absence of cells and cell elements while mechanical properties of ECM were preserved. Conclusion. The developed protocol of esophagus decellularization is a potential basis for obtaining of tissue-engineered esophagus scaffold constructions.

Integration of metabolomics, transcriptomics, and microRNA expression profiling reveals a miR-143-HK2-glucose network underlying zinc-deficiency-associated esophageal neoplasia.

Esophageal squamous cell carcinoma (ESCC) in humans is a deadly disease associated with dietary zinc (Zn)-deficiency. In the rat esophagus, Zn-deficiency induces cell proliferation, alters mRNA and microRNA gene expression, and promotes ESCC. We investigated whether Zn-deficiency alters cell metabolism by evaluating metabolomic profiles of esophageal epithelia from Zn-deficient and replenished rats vs sufficient rats, using untargeted gas chromatography time-of-flight mass spectrometry (n = 8/group). The Zn-deficient proliferative esophagus exhibits a distinct metabolic profile with glucose down 153-fold and lactic acid up 1.7-fold (P < 0.0001), indicating aerobic glycolysis (the “Warburg effect”), a hallmark of cancer cells. Zn-replenishment rapidly increases glucose content, restores deregulated metabolites to control levels, and reverses the hyperplastic phenotype. Integration of metabolomics and our reported transcriptomic data for this tissue unveils a link between glucose down-regulation and overexpression of HK2, an enzyme that catalyzes the first step of glycolysis and is overexpressed in cancer cells. Searching our published microRNA profile, we find that the tumor-suppressor miR-143, a negative regulator of HK2, is down-regulated in Zn-deficient esophagus. Using in situ hybridization and immunohistochemical analysis, the inverse correlation between miR-143 down-regulation and HK2 overexpression is documented in hyperplastic Zn-deficient esophagus, archived ESCC-bearing Zn-deficient esophagus, and human ESCC tissues. Thus, to sustain uncontrolled cell proliferation, Zn-deficiency reprograms glucose metabolism by modulating expression of miR-143 and its target HK2. Our work provides new insight into critical roles of Zn in ESCC development and prevention.