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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Quercetin and ibuprofen combination displayed anti-inflammatory effects and also extenuates the enteric neurons damage of arthritic rats.

This study aimed to investigate the antioxidant and anti-inflammatory properties of quercetin on the cellular components of the Enteric Nervous System in the ileum of rats with arthritis. Rats were distributed into five groups: control (C), arthritic (AIA), arthritic treated with ibuprofen (AI), arthritic treated with quercetin (AQ) and arthritic treated with both ibuprofen and quercetin (AIQ). The ileum was processed for immunohistochemical techniques for HuC/D, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. Measurements in histological sections, chemiluminescence assays, and total antioxidant capacity were also performed. Rheumatoid arthritis resulted in a decrease in neuronal density, yet neuroplasticity mechanisms were evident through observed changes in varicosities size and neuronal area compared to the control group. Reduced paw edema and neuroprotective effects were predominantly noted in both plexuses, as evidenced by the increased density preservation of HuC/D-IR neurons in the AIQ group. The increase of lipoperoxidation levels and paw edema volume in the AQ group was observed compared to the arthritic, whereas the AIQ group mainly showed similar results to those observed in the control. The enteropathy associated with arthritis proved to be significant in the field of gastroenterology, and the combination of quercetin and ibuprofen demonstrated promising anti-inflammatory and neuroprotective effects.

From diversity to disease: unravelling the role of enteric glial cells.

Enteric glial cells (EGCs) are an essential component of the enteric nervous system (ENS) and play key roles in gastrointestinal development, homeostasis, and disease. Derived from neural crest cells, EGCs undergo complex differentiation processes regulated by various signalling pathways. Being among the most dynamic cells of the digestive system, EGCs react to cues in their surrounding microenvironment and communicate with various cell types and systems within the gut. Morphological studies and recent single cell RNA sequencing studies have unveiled heterogeneity among EGC populations with implications for regional functions and roles in diseases. In gastrointestinal disorders, including inflammatory bowel disease (IBD), infections and cancer, EGCs modulate neuroplasticity, immune responses and tumorigenesis. Recent evidence suggests that EGCs respond plastically to the microenvironmental cues, adapting their phenotype and functions in disease states and taking on a crucial role. They exhibit molecular abnormalities and alter communication with other intestinal cell types, underscoring their therapeutic potential as targets. This review delves into the multifaceted roles of EGCs, particularly emphasizing their interactions with various cell types in the gut and their significant contributions to gastrointestinal disorders. Understanding the complex roles of EGCs in gastrointestinal physiology and pathology will be crucial for the development of novel therapeutic strategies for gastrointestinal disorders.

[The gut, a whistleblower, in the early stages of Parkinson's disease]. / L'intestin, lanceur d'alerte, dans les prémices de la maladie de Parkinson.

The enteric nervous system (ENS), often called the "second brain", plays a crucial role in regulating digestive functions. Dysfunctions of the ENS are associated with several diseases such as Parkinson's disease. Recent studies suggest that early digestive disorders, notably chronic constipation, may be early signs of this neurodegenerative disease. Three-dimensional imaging of the ENS offers new insights into early diagnosis, in particular through the analysis of intestinal biopsies. This new research axis raises questions about the intestinal cause of Parkinson's disease, and opens the door to a better understanding and earlier treatment of this disease.

Title: L'intestin, lanceur d'alerte, dans les prémices de la maladie de Parkinson. Abstract: Le système nerveux entérique (SNE), souvent qualifié de « deuxième cerveau ¼, joue un rôle crucial dans la régulation des fonctions digestives. Des dysfonctionnements du SNE sont associés à diverses maladies telles que la maladie de Parkinson. Des études récentes suggèrent que les troubles digestifs précoces, notamment la constipation chronique, pourraient être des signes avant-coureurs de cette maladie neurodégénérative. L'imagerie tridimensionnelle du SNE offre de nouvelles perspectives pour un diagnostic précoce via notamment l'analyse de biopsies intestinales. Ce nouvel axe de recherche soulève des questions sur l'origine intestinale de la maladie de Parkinson et ouvre la porte à une meilleure compréhension et une prise en charge anticipée de cette maladie.

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Enteric Nervous System Striped Patterning and Disease: Unexplored Pathophysiology.

The enteric nervous system (ENS) controls gastrointestinal (GI) motility, and defects in ENS development underlie pediatric GI motility disorders. In disorders such as Hirschsprung's disease (HSCR), pediatric intestinal pseudo-obstruction (PIPO), and intestinal neuronal dysplasia type B (INDB), ENS structure is altered with noted decreased neuronal density in HSCR and reports of increased neuronal density in PIPO and INDB. The developmental origin of these structural deficits is not fully understood. Here, we review the current understanding of ENS development and pediatric GI motility disorders incorporating new data on ENS structure. In particular, emerging evidence demonstrates that enteric neurons are patterned into circumferential stripes along the longitudinal axis of the intestine during mouse and human development. This novel understanding of ENS structure proposes new questions about the pathophysiology of pediatric GI motility disorders. If the ENS is organized into stripes, could the observed changes in enteric neuron density in HSCR, PIPO, and INDB represent differences in the distribution of enteric neuronal stripes? We review mechanisms of striped patterning from other biological systems and propose how defects in striped ENS patterning could explain structural deficits observed in pediatric GI motility disorders.

Brain-first vs. body-first Parkinson's disease: An update on recent evidence.

We recently proposed a new disease model of Parkinson's disease - the a-Synuclein Origin site and Connectome model. The model posits that the initial pathology starts either in the olfactory bulb or amygdala leading to a brain-first subtype, or in the enteric nervous system leading to a body-first subtype. These subtypes should be distinguishable early in the disease course on a range of imaging, clinical, and neuropathological markers. Here, we review recent original human studies, which tested the predictions of the model. Molecular imaging studies were generally in agreement with the model, whereas structural imaging studies, such as MRI volumetry, showed conflicting findings. Most large-scale clinical studies were supportive, reporting clustering of relevant markers of the body-first subtype, including REM-sleep behavior disorder, constipation, autonomic dysfunction, neuropsychiatric symptoms, and cognitive impairment. Finally, studies of a-synuclein deposition in antemortem and postmortem tissues revealed distribution of pathology, which generally supports the model.

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System.

BACKGROUND & AIMS: Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system. METHODS: To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. RESULTS: Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium-induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia. CONCLUSIONS: Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis.

BACKGROUND & AIMS: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Inhibiting the activation of enteric glial cells alleviates intestinal inflammation and comorbid anxiety- and depressive-like behaviors in the ulcerative colitis mice.

Ulcerative colitis (UC) is a common inflammatory bowel disease with a complex origin in clinical settings. It is frequently accompanied by negative emotional responses, including anxiety and depression. Enteric glial cells (EGCs) are important components of the gut-brain axis and are involved in the development of the enteric nervous system (ENS), intestinal neuroimmune, and regulation of intestinal motor functions. Since there is limited research encompassing the regulatory function of EGCs in anxiety- and depression-like behaviors induced by UC, this study aims to reveal their regulatory role in such behaviors and associated intestinal inflammation. This study applied morphological, molecular biological, and behavioral methods to observe the morphological and functional changes of EGCs in UC mice. The results indicated a significant activation of EGCs in the ENS of dextran sodium sulfate -induced UC mice. This activation was evidenced by morphological alterations, such as elongation or terminal swelling of processes. Besides EGCs activation, UC mice exhibited significantly elevated expression levels of pro-inflammatory cytokines in the peripheral blood, accompanied by anxiety- and depression-like behaviors. The inhibition of EGCs activity within the ENS can ameliorate the anxiety- and depression-like behaviors caused by UC. Our data suggest that UC and its resulting behaviors may be related to the activation of EGCs within the ENS. Moreover, the modulation of intestinal inflammation through inhibition of EGCs activation emerges as a promising clinical approach for alleviating UC-induced anxiety- and depression-like behaviors.

Tributyltin (TBT) toxicity: Effects on enteric neuronal plasticity and intestinal barrier of rats' duodenum.

Tributyltin (TBT) is a biocide used in the formulation of antifouling paints and it is highly harmful. Despite the ban, the compound persists in the environment, contaminating marine foodstuffs and household products. Therefore, considering the route of exposure to the contaminant, the gastrointestinal tract (GIT) acts as an important barrier against harmful substances and is a potential biomarker for understanding the consequences of these agents. This work aimed to evaluate histological and neuronal alterations in the duodenum of male Wistar rats that received 20 ng/g TBT and 600 ng/g via gavage for 30 consecutive days. After the experimental period, the animals were euthanized, and the duodenum was intended for neuronal histochemistry (total and metabolically active populations) and histological routine (morphometry and histopathology). The results showed more severe changes in neuronal density and intestinal morphometry in rats exposed to 20 ng/g, such as total neuronal density decrease and reduction of intestinal layers. In rats exposed to 600 ng/g of TBT, it was possible to observe only an increase in intraepithelial lymphocytes. We conclude that TBT can be more harmful to intestinal homeostasis when consumed in lower concentrations.

Does Parkinson's disease start in the gut? / A doença de Parkinson começa no intestino?

ABSTRACT Current understanding of the pathophysiology of Parkinson's disease suggests a key role of the accumulation of alpha-synuclein in the pathogenesis. This critical review highlights major landmarks, hypotheses and controversies about the origin and progression of synucleinopathy in Parkinson's disease, leading to an updated review of evidence suggesting the enteric nervous system might be the starting point for the whole process. Although accumulating and compelling evidence favors this theory, the remaining knowledge gaps are important points for future studies.

RESUMO O atual entendimento sobre a fisiopatologia da doença de Parkinson (DP) sugere um papel central do acúmulo de alfa-sinucleína na patogenia da DP Esta revisão crítica revisita marcos, teorias e controvérsias a respeito da origem e progressão da sinucleinopatia, apresentando uma atualização das principais evidências sugerindo que o sistema nervoso entérico seria o local inicial deste processo. Apesar das evidências a favor desta teoria serem crescentes e instigantes, as lacunas de conhecimento a este respeito são importantes pontos para estudos futuros.

Do lesions of the enteric nervous system occur following intestinal ischemia/reperfusion?

PURPOSE: To evaluate tissue lesions, especially those of the intestinal innervation, in an excluded jejunal loop subjected to ischemia and reperfusion in rats. METHODS: To evaluate the role of ischemia and reperfusion lesions in an excluded intestinal loop, four groups of 20 rats were set up: control group (GCEI7) and three experimental groups (GIREI7, GIREI14 and GIREI28). They were all subjected to exclusion of an intestinal segment of six centimeters in length, at a distance of 10 centimeters from the Treitz angle. The 60 animals in the three experimental groups were additionally subjected to ischemia of the vascular pedicle for 30 minutes. The control group and the experimental group GIREI7 were evaluated on the 7th day after the operation. The groups GIREI14 and GIREI28 (which also underwent ischemia) were utilized to evaluate the evolution of the lesion over time, on the 14th and 28th days after the operation, respectively. From the intestinal excluded loop, we take one ring of 0,5 cm distal and proximal, that were fixed in formaline 10 percent solution in order to do histological (HE) and immuno-hystochemial (PS-100) evaluation (enteric nervous system.) The distal loop was exteriorized in stoma and the proximal part closed with polipropilene 6-0. RESULTS: It was observed a decrease in the number of ganglionic cells in the myenteric plexus in the group subjected to ischemia and reperfusion (GIREI7), in relation to the control group (GCEI7) at the 7th post-operative day (Mann-Whitney test: p = 0.0173 *. Comparing the numbers of ganglionic cells in the myenteric plexus before and after jejunal loop exclusion GCEI7 - (Wilcoxon test: p = 0.0577). GIREI7 - Comparing the numbers of ganglionic cells in the myenteric plexus before and after ischemia (*p = 0.0399). Comparing the percentage variations in ganglionic cells in the myenteric plexus on the 7th, 14th and 28th days after the procedure, in the groups GIREI7, GIREI14 and GIREI28,...

OBJETIVO: Avaliar lesões teciduais, especialmente aquelas da inervação intestinal em alça jejunal excluída submetida à isquemia e reperfusão em ratos. MÉTODOS: Para avaliar o papel da isquemia e reperfusão nas lesões em uma alça intestinal exclusa, quatro grupos de 20 ratos foram criados: Grupo controle (GCE17) e 3 Grupos experimentais (GIRE!7, GIREI14) e GIREI28) Todos foram submetidos à exclusão de um segmento intestinal de seis centímetros de extensão, a 10 centímetros do ângulo de Treitz . Os 60 animais dos 3 grupos experimentais foram também submetidos a isquemia do pedículo vascular por 30 minutos.O grupo controle e o grupo experimental GIREI7 foram avaliados no 7°. Dia após a operação. Os grupos GIREI14 e GIREI28 também submetidos à isquemia, foram utilizados para avaliar a evolução da lesão com o passar do tempo, no 14°. e 28°. dias respectivamente. Do segmento intestinal excluído do trânsito, foi retirada uma amostra de 0,5 cm em cada extremidade, proximal e distal, as quais foram fixadas em solução de formol 10 por cento para posterior avaliação histológica, com HE e imuno histoquímica pela proteína PS-100 para avaliação do sistema nervoso entérico. A luz distal da alça isolada foi estomizada e a proximal fechada com pontos de prolene 6-0. Esses dados foram analisados estatisticamente. RESULTADOS: Observamos uma diminuição do número de células ganglionares no plexo mioentérico do grupo submetido à isquemia e reperfusão (GIREI7) em relação ao grupo controle (GCEI7). Mann-Whitney: p=0,0173*. Comparando a variação percentual das células ganglionares do plexo mioentérico no 7°, 14° e 28° dia após procedimento nos grupos GIREI7, GIREI14 E GIREI28 observamos que não houve alterações significantes. Kruskal-Wallis p=0,6501. CONCLUSÃO: Houve uma diminuição das células ganglionares nos plexos mioentéricos devido à isquemia e reperfusão, não havendo recuperação no período pós-operatório tardio.

Study of the density of ganglion cells in the terminal bowel of rats with anorectal malformations

PURPOSE: To study the ganglion cells (GC) in the terminal bowel of rats with ethylenethiourea (ETU) induced anorectal malformations (ARM). METHODS: The animals were divided into three groups: Group A - normal fetuses from pregnant rats that were not administered ETU; Group B - fetuses without ARM born from pregnant rats that were administered ETU and Group C - fetuses with ARM born from pregnant rats that received ETU. ETU was administered on the 11th day of pregnancy at the dose of 125 mg/kg body weight by gastric gavage. The rats had cesarean section on the 21st day of gestation. The fetuses’ terminal bowel tissue was analyzed by immunohistochemistry to demonstrate ganglion cells. RESULTS: Statistically significant differences were found between groups A, B and C regarding ganglion cell densities. Group A had the highest cell density, followed by Group B and the lowest density was found in Group C. CONCLUSION: Ganglion cell densities are decreased in the terminal bowel of rats with ARM.

OBJETIVO: Estudar as células ganglionares (CG) no intestino terminal de ratos portadores de anomalia anorretal (AAR) induzida pela etilenotiouréia (ETU). MÉTODOS: Os animais foram distribuídos em três grupos: Grupo A - fetos normais, obtidos de ratas grávidas às quais não foi administrada ETU; Grupo B - fetos não portadores de AAR obtidos de ratas grávidas às quais foi administrada ETU e Grupo C - fetos portadores de AAR obtidos de ratas grávidas às quais foi administrada ETU. A ETU foi administrada no décimo primeiro dia de gestação na dose de 125 mg/Kg, por gavagem. As ratas foram submetidas à laparotomia e histerotomia para retirada dos fetos no vigésimo primeiro dia de gestação. O intestino terminal dos fetos foi retirado e analisado por imunohistoquímica para pesquisa de CG. RESULTADOS: Foram encontradas diferenças estatisticamente significantes entre os grupos A, B e C quanto à densidade de CG. O grupo A apresentou a maior densidade, seguida pelo grupo B, e a menor densidade foi encontrada no Grupo C. CONCLUSÃO: Existe uma menor densidade de CG no intestino terminal de ratos portadores de AAR.

Morphoquantitative evaluation of the duodenal myenteric neurona} population in rats fed with hypoproteic ration

The purpose of this work was to analyze the morphoquantitative behavior of the neurons of the myenteric plexus, as well as the morphometry of the duodenal wall, in adult rats fed with normoproteic (22%) and hypoproteic (8%) rations, killed at the age of 345 days. For neuronal assessments duodenal wholernounts stained with the Giemsa method were used, and for the evaluation of the duodenal wall routine histological processing and staining with Hematoxilin-Eosin were employed. The means of the number of neurons in 80 microscopic fields (12.72 mm2) and of the size of the neuronal cell bodies did not reveal statistically significant differences between the groups, but there was a greater incidence of large neurons in the protein restriction group (RP). The duodenum was markedly smaller in the RP group and, although there was no difference in the thickness of its wall, the mucosa was larger and the muscular layer was smaller in group RP. It was concluded that the neuronal and non-neuronal components of the duodenum adjust themselves to the nutritional condition, assuring the maintenance of their functions