Myenteric Research Articles

Esophageal Motility Disorders: How Does the Barium Swallow Correlate With Manometry?

Achalasia is defined as dysfunction of the esophageal wall myenteric plexus, which causes symptoms of dysphagia. While manometry is typically regarded as the gold standard for diagnosing and confirming achalasia, other imaging modalities such as barium swallow and upper endoscopy are often obtained initially. The barium swallow study can be a supportive or confirmatory test, whereas the upper endoscopy is typically used to rule out pseudo-achalasia. Additionally, barium swallow is an imaging modality of choice in resource-limited settings. A standardized approach for categorizing motility disorders is the Chicago Classification. This is a newer classification of achalasia based on high-resolution manometry. However, the role of barium contrast studies does not seem to have been evaluated in the context of the Chicago Classification of the three achalasia subtypes, suggesting an underappreciated role of fluoroscopy in the diagnostic evaluation of achalasia subtypes.

Morphometric Changes of the Myenteric Plexus in the Colon of the Mice-D-Galactose Ageing Model

Ageing-related diseases are paramount for understanding the socioeconomic impact of the postmodern society. Neuronal degeneration is a known phenomenon in ageing. The D-galactose model of accelerated ageing is widely used in age-associated neuronal cell death because of the oxidative stress it induces, which correlates to reduced cognitive performance and robustly detectable neuronal shrinkage in the CNS. This study aims at demonstrating the morphological changes of neurons of the myenteric plexus in mice treated with D-galactose. D-galactose was given orally with drinking water, resulting in an average dose of 500 mg/kg daily for six weeks. In the D-galactose accelerated ageing model, a significant size reduction of the neuronal perikarya of the myenteric plexus was observed all along the large intestine. The average soma area at the caecum in the control group was 305 µm2, which was reduced by almost 20% in the ageing model. In contrast, at the level of the proximal colon in the D-galactose ageing model, the average area of the neuronal soma was 280 µm2, a reduction of 30%. The most significant reduction has been observed at the level of the distal colon, where the neuronal soma was reduced by 40%. There is a significant reduction of the area of the neuronal bodies in the myenteric plexus among different parts of the mice's large intestine in the accelerated ageing model. It can be inferred that D-galactose-induced morphological changes in the myenteric plexus may be related to the increased gastrointestinal motility dysfunction with ageing.

QbD approach for developing Fast-dissolving Tablets containing Loperamide hydrochloride using Super Disintegrant Blends and Subliming Materials

Mouth dissolving pills, which dissolve or disintegrate quickly on the tongue or buccal cavity, have been developed in response to the growing demand for more patient-compliant dosage forms. It is utilized to improve bioavailability by reducing dose frequency in order to reduce adverse effects and make tablets with high first-pass metabolism more cost-effective. The most commonly used drug for diarrhoea is loperamide hydrochloride. It is an opioid receptor agonist that targets the mu opioid receptors in the large intestines of the myenteric plexus. It specifically works by lowering myenteric plexus activity, which lowers the motility of the circular and longitudinal smooth muscles of the intestinal wall. It is a synthetic anti-diarrheal that is used to treat the symptoms of inflammatory bowel disease-related chronic diarrhoea as well as acute, non-specific diarrhoea. Due to substantial hepatic first-pass metabolism, loperamide hydrochloride has a very limited systemic bioavailability. Therefore, the primary goal of the study was to develop Loperamide hydrochloride orally disintegrating tablets to obtain a better breakdown rate, enhance the drug's bioavailability, and provide very rapid relief from diarrhoea. The pre-compression parameters, such as bulk density, compressibility, angle of repose, etc., for orally disintegrating tablets manufactured by direct compression and using three super disintegrants, croscarmellose sodium (CCS), Microcrystalline cellulose (MCC), and sodium starch glycolate (SSG), were prepared and assessed. The manufactured batches of tablets passed tests for satisfactory results. The highest amount of medicine was released at all-time intervals in the optimised formulation, which also displayed a favourable release profile.

Protective effect of oxytocin on vincristine-induced gastrointestinal dysmotility in mice.

Aims: Vincristine (VCR), an antineoplastic drug, induces peripheral neuropathy characterized by nerve damage, limiting its use and reducing the quality of life of patients. VCR causes myenteric neuron damage, inhibits gastrointestinal motility, and results in constipation or paralytic ileus in patients. Oxytocin (OT) is an endogenous neuropeptide produced by the enteric nerve system, which regulates gastrointestinal motility and exerts neuroprotective effects. This study aimed to investigate whether OT can improve VCR-induced gastrointestinal dysmotility and evaluate the underlying mechanism. Methods: Mice were injected either with saline or VCR (0.1mg/kg/d, i. p.) for 14 days, and OT (0.1mg/kg/d, i.p.) was applied 1h before each VCR injection. Gastrointestinal transit and the contractile activity of the isolated colonic segments were assessed. The concentration of OT in plasma was measured using ELISA. Immunofluorescence staining was performed to analyze myenteric neurons and reactive oxygen species (ROS) levels. Furthermore, the indicators of oxidative stress were detected. The protein expressions of Nrf2, ERK1/2, P-ERK1/2, p38, and P-p38 in the colon were tested using Western blot. Results: VCR reduced gastrointestinal transit and the responses of isolated colonic segments to electrical field stimulation and decreased the amount of neurons. Furthermore, VCR reduced neuronal nitric oxide synthase and choline acetyltransferase immunopositive neurons in the colonic myenteric nerve plexus. VCR increased the concentration of OT in plasma. Exogenous OT pretreatment ameliorated the inhibition of gastrointestinal motility and the injury of myenteric neurons caused by VCR. OT pretreatment also prevented the decrease of superoxide dismutase activity, glutathione content, total antioxidative capacity, and Nrf2 expression, the increase of ROS levels, and the phosphorylation of ERK1/2 and p38 MAPK following VCR treatment. Conclusion: Our results suggest that OT pretreatment can protect enteric neurons from VCR-induced injury by inhibiting oxidative stress and MAPK pathways (ERK1/2, p38). This may be the underlying mechanism by which it alleviates gastrointestinal dysmotility.

The depth of perineural invasion is an independent prognostic factor for stage II colorectal cancer.

Perineural invasion (PNI) is the invasion of nerves by cancer cells and is associated with poor survival in stage II colorectal cancer. However, PNI can be further subdivided according to the depth of invasion, and the depth of PNI has not been clearly linked to prognosis. This study aimed to assess the prognostic value of different depths of PNI in stage II colorectal cancer. We defined PNI in the submucosal plexus and myenteric plexus as superficial perineural invasion (sup-PNI) and PNI in the subserous plexus as deep perineural invasion (deep-PNI). Patients were divided into three groups based on the depth of PNI: sup-PNI, deep-PNI and non-PNI. Then, univariate and multivariate Cox regression analyses were conducted to evaluate the role of PNI in the prognosis of stage II colorectal cancer. This study enrolled 3508 patients with stage II colorectal cancer who underwent resection for primary colorectal lesions between January 2013 and September 2019. Clinicopathological features, including elevated carcinoembryonic antigen (CEA) levels, T4 stage, poor differentiation, deficient DNA mismatch repair (dMMR), and vascular invasion, were correlated with deep-PNI. Multivariate analyses revealed that deep-PNI was associated with worse overall survival (OS; hazard ratio [HR], 3.546; 95% confidence interval [CI], 2.307-5.449; P < 0.001) and disease-free survival (DFS; HR, 2.921; 95% CI, 2.032-4.198; P < 0.001), compared with non-PNI. Conversely, no significant difference in OS or DFS was observed between the sup-PNI and non-PNI groups in multivariate analyses. The study demonstrated that the depth of PNI was an independent prognostic factor for patients with stage II colorectal cancer, and patients with deep PNI had a worse prognosis. Thus, patients with PNI require further subdivision according to the depth of invasion.

Inhibition of calcium-sensing receptor by its antagonist promotes gastrointestinal motility in a Parkinson’s disease mouse model

BackgroundThe Calcium-sensing receptor (CaSR) participates in the regulation of gastrointestinal (GI) motility under normal conditions and might be involved in the regulation of GI dysmotility in patients with Parkinson's disease (PD). MethodsCaSR antagonist-NPS-2143 was applied in in vivo and ex vivo experiments to study the effect and underlying mechanisms of CaSR inhibition on GI dysmotility in the MPTP-induced PD mouse model. FindingsOral intake of NPS-2143 promoted GI motility in PD mice as shown by the increased gastric emptying rate and shortened whole gut transit time together with improved weight and water content in the feces of PD mice, and the lack of influence on normal mice. Meanwhile, the number of cholinergic neurons, the proportion of serotonergic neurons, as well as the levels of acetylcholine and serotonin increased, but the numbers of nitrergic and tyrosine hydroxylase immunoreactive neurons, and the levels of nitric oxide synthase and dopamine decreased in the myenteric plexus in the gastric antrum and colon of PD mice in response to NPS-2143 treatment. Furthermore, the numbers of c-fos positive neurons in the nucleus tractus solitarius (NTS) and cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) increased in NPS-2143 treated PD mice, suggesting the involvement of both the enteric (ENS) and central (CNS) nervous systems. However, ex vivo results showed that NPS-2143 directly inhibited the contractility of antral and colonic strips in PD mice via a non-ENS mediated mechanism. Further studies revealed that NPS-2143 directly inhibited the voltage gated Ca2+ channels, which might, at least in part, explain its direct inhibitory effects on the GI muscle strips. InterpretationCaSR inhibition by its antagonist ameliorated GI dysmotility in PD mice via coordinated neuronal regulation by both ENS and CNS in vivo, although the direct effects of CaSR inhibition on GI muscle strips were suppressive.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung’s Disease

Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-μm2× 500-μm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

The role of galanin in the progression and prognosis of colorectal cancer: the unfinished story.

The paper presents a summary of immunohistochemical (IHC) and biochemical investigations on the presence of galanin (Gal), one of the neuropeptides abundant in the enteric nervous systems, and three types of its receptors (GalR1-3) in colorectal cancer (CRC) tissue and non-involved colon wall and their associations with clinical-pathological data of the CRC patients. We were the first to morphologically demonstrate the presence of endogenous Gal in CRC sections and measure its content in homogenates of tumor tissue and dissected compartments of unchanged colon wall. The prominent atrophy of myenteric plexuses displaying Gal immunoreactivity (Gal-Ir) located close to the tumor invasion was found to be accompanied by higher Gal content in the tumor-adjacent muscularis externa than in tumor-distant tissue. In further studies for the first time, we demonstrated by the IHC technique the presence of the GalR1-3 receptors in the CRC tumors and the colon mucosa and found that higher GalR3-Ir in the tumor tissue correlated with longer overall survival of CRC patients. Furthermore, we discovered that lower GalR1 expression in submucosal plexuses located near the tumor correlated with a better prognosis in patients with CRC. These findings suggest that GalR1 could be considered as a novel therapeutic target in CRC. In conclusion, our morphological investigations provided novel data documenting the involvement of Gal and its receptors in the progression of CRC and showed the usefulness of the IHC technique for the prognosis of CRC patients.

FABP2 is Involved in Intestinal α-Synuclein Pathologies.

Recently, the hypothesis that pathological α-Synuclein propagates from the gut to the brain has gained attention. Although results from animal studies support this hypothesis, the specific mechanism remains unclear. This study focused on the intestinal fatty acid-binding protein (FABP2), which is one of the subtypes of fatty acid binding proteins localizing in the gut, with the hypothesis that FABP2 is involved in the gut-to-brain propagation of α-synuclein. The aim of this study was to clarify the pathological significance of FABP2 in the pathogenesis and progression of synucleinopathy. We examined the relationship between FABP2 and α-Synuclein in the uptake of α-Synuclein into enteric neurons using primary cultured neurons derived from mouse small intestinal myenteric plexus. We also quantified disease-related protein concentrations in the plasma of patients with synucleinopathy and related diseases, and analyzed the relationship between plasma FABP2 level and progression of the disease. Experiments on α-Synuclein uptake in primary cultured enteric neurons showed that following uptake, α-Synuclein was concentrated in areas where FABP2 was localized. Moreover, analysis of the plasma protein levels of patients with Parkinson's disease revealed that the plasma FABP2 and α-Synuclein levels fluctuate with disease duration. The FABP2/α-Synuclein ratio fluctuated more markedly than either FABP2 or α-Synuclein alone, depending on the duration of disease, indicating a higher discriminant ability of early Parkinson's disease patients from healthy patients. These results suggest that FABP2 potentially contributes to the pathogenesis and progression of α-synucleinopathies. Thus, FABP2 is an important molecule that has the potential to elucidate the consistent mechanisms that lead from the prodromal phase to the onset and subsequent progression of synucleinopathies.

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Pathological investigation of high pathogenicity avian influenza H5N8 in captive houbara bustards (Chlamydotis undulata), the United Arab Emirates 2020

At the end of 2020, an outbreak of HPAI H5N8 was registered in captive African houbara bustards (Chlamydotis undulata) in the United Arab Emirates. In order to better understand the pathobiology of this viral infection in bustards, a comprehensive pathological characterization was performed. A total of six birds were selected for necropsy, histopathology, immunohistochemistry, RNAscope in situ hybridization and RT-qPCR and nanopore sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue blocks. Gross lesions included mottled and/or hemorrhagic pancreas, spleen and liver and fibrinous deposits on air sacs and intestine. Necrotizing pancreatitis, splenitis and concurrent vasculitis, hepatitis and fibrino-heterophilic peritonitis were identified, microscopically. Viral antigens (nucleoprotein) and RNAs (matrix gene) were both detected within necro-inflammatory foci, parenchymal cells, stromal cells and endothelial cells of affected organs, including the myenteric plexus. Molecular analysis of FFPE blocks successfully detected HPAI H5N8, further confirming its involvement in the lesions observed. In conclusion, HPAI H5N8 in African houbara bustards results in hyperacute/acute forms exhibiting marked pantropism, endotheliotropism and neurotropism. In addition, our findings support the use of FFPE tissues for molecular studies of poorly characterized pathogens in exotic and endangered species, when availability of samples is limited.

Ameliorating effects of transcutaneous auricular vagus nerve stimulation on a mouse model of constipation-predominant irritable bowel syndrome

Limited treatment options have been shown to alter the natural course of constipation-predominant irritable bowel syndrome (IBS-C). Therefore, safer and more effective approaches are urgently needed. We investigated the effects of transcutaneous auricular vagus nerve stimulation (taVNS) in a mouse model of IBS-C. In the current study, C57BL/6 mice were randomly divided into normal control, IBS-C model control, sham-electrostimulation (sham-ES), taVNS, and drug treatment groups. The effects of taVNS on fecal pellet number, fecal water content, and gastrointestinal transit were evaluated in IBS-C model mice. We assessed the effect of taVNS on visceral hypersensitivity using the colorectal distention test. 16S rRNA sequencing was used to analyze the fecal microbiota of the experimental groups. First, we found that taVNS increased fecal pellet number, fecal water content, and gastrointestinal transit in IBS-C model mice compared with the sham-ES group. Second, taVNS significantly decreased the abdominal withdrawal reflex (AWR) score compared with the sham-ES group, thus relieving visceral hyperalgesia. Third, the gut microbiota outcomes showed that taVNS restored Lactobacillus abundance while increasing Bifidobacterium probiotic abundance at the genus level. Notably, taVNS increased the number of c-kit-positive interstitial cells of Cajal (ICC) in the myenteric plexus region in IBS-C mice compared with the sham-ES group. Therefore, our study indicated that taVNS effectively ameliorated IBS-C in the gut microbiota and ICC.

A8 LUMINAL SHORT CHAIN FATTY ACIDS INCREASE ACTIVITY IN COLONIC MYENTERIC PLEXUS NEURONS IN MICE

Abstract Background The enteric nervous system (ENS) plays a critical role in the control of gastrointestinal functions. Short chain fatty acids (SCFAs) are the products of non-digestible dietary fiber fermentation by gut microbial metabolism and are a primary energy source for enterocytes. However, the impact of SCFAs on the ENS is not well understood. Aims We aimed to test whether SCFAs affect enteric neuronal function using a novel intact preparation of the colon. Methods Intact segments of the colon from mice expressing a genetically encoded fluorescent calcium (Ca2+) reporter specifically in all enteric neurons (Wnt-1-GCaMP6 mice) or in intrinsic primary afferent neurons (IPANs, Calb1-GCaMP6 mice) were bathed and luminally perfused with Krebs solution. Contractility was inhibited by nicardipine (3 µM) and scopolamine (1 µM). The duration and intensity of neuronal intracellular Ca2+ fluorescence responses to SCFA were visualized by live-cell confocal microscopy and were analyzed using Imaris™ software. The SCFAs were luminally perfused at a constant rate of 10 mL/h for 3 min. NaCl was modified in Krebs solution to correct for the osmolarity of SCFAs. Results In the myenteric plexus of Wnt-1-GCaMP6 mice, SCFAs (acetate, 60 mM; propionate, 20 mM or butyrate, 20 mM) caused a rapid and sustained increase in Ca2+ fluorescence (26.8±5.9, 25.8±1.7, and 27.4±5.9 min, respectively). The peak Ca2+ responses were 92.5%± 28.3%, 122.6%±21.5% and 111.7%± 23.0%, respectively. The combination of these three SCFAs increased Ca2+ fluorescence for 20.5±6.3 min and potentiated levels of Ca2+ fluorescence to 125.1%±21.6% above baseline. The percentages of neurons responding to SCFAs were 85.7%, 94.4%, 97.7% and 93.3 % for acetate, propionate, butyrate and a combination of SCFAs, respectively. In Calb1-GCaMP6 mice, IPANs showed a similar pattern of Ca2+ responses and a proportion of neurons that responded to the SCFAs. Following acetate, propionate, butyrate and combination of SCFAs administration, fluorescence was increased for 13.5±1.2, 20.6±2.7, 24.9±2.2 and 46.1±4.6 min, by a magnitude of 404.3%±153.7%, 129.0%±0.4%, 73.9%±18.8% and 717%±267.0% above baseline and the percentages of neuronal response were 100.0%, 100.0%, 100.0% and 87.5 %, respectively. Conclusions We show that SCFAs activate neurons of the myenteric plexus including IPANs. These studies suggest that some of the actions of SCFAs are mediated by the ENS, but further studies are needed to identify the underlying mechanisms of action of SCFAs in the myenteric plexus. Funding Agencies CAG, CIHRTRIANGLE Canada

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

5-Hydroxytryptamine 4 Receptor Agonist Attenuates Diabetic Enteric Neuropathy through Inhibition of the Receptor-Interacting Protein Kinase 3 Pathway

Necroptosis, considered as a form of programmed cell death, contributes to the neural loss. The 5-hydroxytryptamine 4 receptor (5-HT4R) is involved in neurogenesis in the enteric nervous system (ENS). However, it is unclear whether the activation of 5-HT4R can alleviate diabetic enteric neuropathy by inhibiting receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis. This study aimed to explore the beneficial effects of 5-HT4R agonist on enteric neuropathy in a mouse model of diabetes and the mechanisms underlying these effects. Diabetes developed neural loss in the colon of mice. 5-HT4Rs localized in submucosal and myenteric plexuses were confirmed. Administration of 5-HT4R agonist attenuated diabetes-induced colonic hypomotility and neural loss of the colon in mice. Remarkably, RIPK3, phosphorylated RIPK3 (pRIPK3) and its downstream target mixed lineage kinase domain-like protein (MLKL), two key proteins regulating necroptosis, were significantly upregulated in the colon of diabetic mice. Treatment with 5-HT4R agonist appeared to inhibit diabetes-induced elevation of RIPK3, pRIPK3 and MLKL in the colon of mice. Diabetes-induced upregulation of MLKL in both the mucosa and muscularis of the colon was prevented by RIPK3 deletion. Moreover, Diabetes-evoked neural loss and delayed colonic transit were significantly inhibited by RIPK3 removal. These findings suggest that activation of 5-HT4R receptors could potentially provide a protective effect against diabetic enteric neuropathy by suppressing RIPK3-mediated necroptosis.

Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

P205 Histologic image-based ensemble model to identify myenteric plexitis and predict endoscopic postoperative recurrence in Crohn’s disease: a multicentre, retrospective study

Abstract Background Myenteric plexitis is correlated with postoperative recurrence of Crohn’s disease (CD) when relying on traditional statistical methods. However, comprehensive assessment of the myenteric plexus remains challenging. This study aimed to develop and validate a deep learning system to predict postoperative recurrence through automatic screening and identification of features of the muscular layer and myenteric plexus. Methods In this study, we retrospectively reviewed 205 CD patients who underwent bowel resection surgery from 2 hospitals. Patients were divided into a training cohort (n=108), an internal validation cohort (n=47) and an external validation cohort (n=50). A total of 190960 patches from 278 whole-slide images of surgical specimens were analysed using ResNet50, and 6144 features were extracted after transfer learning. Spearman correlation analysis, LASSO logistic regression and the interclass correlation coefficient test were utilized for feature selection. We used five robust algorithms to construct classification models. The performances of the models were evaluated by the area under the receiver operating characteristic curve (AUC) in three cohorts. Results The stacking model achieved satisfactory accuracy in predicting postoperative recurrence of CD in the training cohort (AUC: 0.980; 95% CI 0.960-0.999), internal validation cohort (AUC: 0.908; 95% CI 0.823-0.992), and external validation cohort (AUC: 0.868; 95% CI 0.761-0.975). The accuracy for identifying the severity of myenteric plexitis was 0.833, 0.745, and 0.694 in the training cohort, internal validation cohort and external validation cohort, respectively. Conclusion Our work initially established an interpretable stacking model based on muscular layer and myenteric plexus features extracted from histologic images to identify the severity of myenteric plexitis and predict postoperative recurrence of CD.

Achalasia alters physiological networks depending on sex

Achalasia is a rare esophageal motility disorder for which the etiology is not fully understood. Evidence suggests that autoimmune inflammatory infiltrates, possibly triggered by a viral infection, may lead to a degeneration of neurons within the myenteric plexus. While the infection is eventually resolved, genetically susceptible individuals may still be at risk of developing achalasia. This study aimed to determine whether immunological and physiological networks differ between male and female patients with achalasia. This cross-sectional study included 189 preoperative achalasia patients and 500 healthy blood donor volunteers. Demographic, clinical, laboratory, immunological, and tissue biomarkers were collected. Male and female participants were evaluated separately to determine the role of sex. Correlation matrices were constructed using bivariate relationships to generate complex inferential networks. These matrices were filtered based on their statistical significance to identify the most relevant relationships between variables. Network topology and node centrality were calculated using tools available in the R programming language. Previous occurrences of chickenpox, measles, and mumps infections have been proposed as potential risk factors for achalasia, with a stronger association observed in females. Principal component analysis (PCA) identified IL-22, Th2, and regulatory B lymphocytes as key variables contributing to the disease. The physiological network topology has the potential to inform whether a localized injury or illness is likely to produce systemic consequences and the resulting clinical presentation. Here we show that immunological involvement in achalasia appears localized in men because of their highly modular physiological network. In contrast, in women the disease becomes systemic because of their robust network with a larger number of inter-cluster linkages.

P440 Myenteric plexitis and active inflammation at surgical margins are high-risk predictors for postoperative clinical recurrence of Crohn’s disease

Abstract Background Preventing postoperative recurrence is an important issue for clinical management of Crohn’s disease (CD). Identification of practical risk factors and an effective prophylactic treatment strategy has been desired for the prevention of postoperative recurrence. Although some of the clinical risk factors have been applied to select candidates for prophylactic treatments, these attempts have not fully prevented postoperative recurrence. In this study, we investigate the histological features at resected margins which predicted postoperative recurrence, and whether the initiation of prophylactic treatments could prevent postoperative recurrence in these cases. Methods We retrospectively reviewed the clinical recurrence rate in CD patients who underwent intestinal resection between January 2019 and December 2021. Clinical recurrence was defined by combination of serological, radiographic, and endoscopic findings or treatment escalation. To assess the histological risk factors for postoperative recurrence, resection margins of the surgical specimens were evaluated for the presence of myenteric plexitis, granulomas, and active inflammation. Each histological feature was evaluated from Grade 0 to 3. We also examined the ability of prophylactic treatments to prevent postoperative recurrence stratified with histological findings related to clinical recurrence rates. Results Of the 141 patients included in this study, 61.9% had plexitis (grade 3:Inflammatory cells at myenteric plexus, 10&amp;gt;HPF) at the proximal margin and 13.5% had active inflammation (grade 3: erosion or ulcer) at the distal margin. Log rank test identified the presence of plexitis (Grade3) (HR:1.84, 95%CI [1.05-3.22], p=0.029) at the proximal margin and active inflammation (Grade3) at the distal margin (HR:1.29, 95%CI [1.05-1.58], p=0.012) as possible risks of clinical recurrence (Figure). Multivariate analysis confirmed that the presence of plexitis (Grade3) at the proximal margin (HR 1.82, 95% CI 1.04-3.21, P=0.038) and active inflammation (Grade 3) at the distal margin (HR 2.31, 95% CI 1.18-4.46, P=0.013) were associated with postoperative clinical recurrence. Prophylactic use of immunomodulators (IM) and biologics did not show a significant reduction of clinical recurrence compared to treatment without IM and/or biologics in patient with histological risk factors (Table). Conclusion Our results indicate that the presence of plexitis in the proximal margin and active inflammation in the distal margin of the resected intestine are risk factors for postoperative recurrence. In these cases, initiation of prophylactic treatments with biologics and/or IM may not prevent the clinical recurrence.

P027 Involvement of PD-L1 in myenteric plexitis associated with postoperative recurrence of Crohn's disease

Abstract Background Despite a growing therapeutic armamentarium, post-operative recurrence (POR) in Crohn’s disease (CD) remains frequent. The presence of myenteric plexitis at the proximal margin of ileocolonic resection has been recognized as a risk factor for early POR. They are defined by the accumulation of immune cells in contact with the ganglia of the myenteric plexus. In preliminary work, we have shown an increase in the number of T lymphocytes (TL) in contact with enteric glial cells (EGC) in patients with CD depending on ICAM-1/LFA-1 pathway. As CD is classically associated with a Th1 immune response, the aim of this work was to study the impact of EGC/CD4+ TL contacts on Th1-type cytokine production (IFNγ and its transcription factor Tbet) and to identify molecules that may be involved in these regulations. Methods In vitro in rats, the impact of EGC/CD4+ TL co-culture on IFNγ production was studied by ELISA and qPCR of the transcription factor Tbet. The expression and regulation of PD-L1, a candidate molecule, was then analyzed in murine and human EGC by qPCR and immunohisto/cytochemistry. Results While EGC alone produced only a small amount of IFNγ, there was a significant reduction in IFNγ production in EGC/CD4+ TL co-cultures compared with CD4+ TL alone. Yet the proportion of CD4+ TL cells capable of producing IFNγ was significantly increased in contact with EGC, as was their survival. In parallel, Tbet was downregulated in CD4+ TL in contact with EGC. These results suggested the involvement of PD-L1, a molecule known to negatively regulate cytokine production (notably IFNγ) and Tbet expression. Expression of PD-L1 mRNA and its protein was demonstrated in murine and human EGC and was upregulated under pro-inflammatory conditions. Analysis of transmural sections at the proximal margin of ileocolonic resection from patients (n=15 controls, n=14 CD without POR, n=15 CD with POR) showed PD-L1 expression in myenteric ganglia that was restricted to EGC (no neuronal expression) (Figure 1A). PD-L1 fluorescence intensity in myenteric ganglia at the proximal resection margin was significantly lower in CD patients with POR than in controls (∆13.5%; p=0.012) (Figure 1B). Conclusion Lower IFNγ production in EGC/CD4+ TL co-cultures associated with reduced Tbet expression by TL in contact with EGC suggest a role of PD-L1 in myenteric plexitis. PD-L1 expression has been demonstrated in murine and human EGC, with significantly lower expression in myenteric ganglia located at the proximal resection margin of CD patients with POR compared with controls. Blocking the ICAM-1/LFA-1 pathway and/or restoring the PD-1/PD-L1 loop could be of interest for developing strategies to prevent and/or treat POR in CD patients.