Gastric and sacral electrical stimulation for motility disorders-A clinical perspective.

BACKGROUND: Electrical stimulation of the gut has been investigated in recent decades with a view to treating various gastro-intestinal motility disorders including, among others, gastric electrical stimulation to relieve nausea and vomiting associated with gastroparesis and sacral neuromodulation to treat fecal incontinence and/or constipation. Although their symptomatic efficacy has been ascertained by randomized controlled trials, their mechanisms of action are not fully understood. PURPOSE: This review summarizes the past year's literature on the mechanisms of action of gut electrical stimulation therapies, including their impact on the gut-brain axis.

Spontaneous enteric nervous system activity generates contractile patterns prior to maturation of gastrointestinal motility.

BACKGROUND: Spontaneous neuronal network activity is essential to the functional maturation of central and peripheral circuits, yet whether this is a feature of enteric nervous system development has yet to be established. Although enteric neurons are known exhibit electrophysiological properties early in embryonic development, no connection has been drawn between this neuronal activity and the development of gastrointestinal (GI) motility patterns. METHODS: We use ex vivo GI motility assays with newly developed unbiased computational analyses to identify GI motility patterns across mouse embryonic development. KEY RESULTS: We find a previously unknown pattern of neurogenic contractions termed "clustered ripples" that arises spontaneously at embryonic day 16.5, an age earlier than any identified mature GI motility patterns. We further show that these contractions are driven by nicotinic cholinergic signaling. CONCLUSIONS & INFERENCES: Clustered ripples are neurogenic contractile activity that arise from spontaneous ENS activity and precede all known forms of neurogenic GI motility. This earliest motility pattern requires nicotinic cholinergic signaling, which may inform pharmacology for enhancing GI motility in preterm infants.

Somatostatin-immunoreactive neurons of the rat gut during the development.

Somatostatin (SST) is a peptide expressed in the peripheral and central nervous systems, as well as in endocrine and immune cells. The aim of the current study is to determine the percentage of SST immunoreactive (IR) neurons and their colocalization with choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and glial fibrillary acidic protein (GFAP) in the myenteric plexus (MP) and submucous plexus (SP) of the small intestine (SI) and large intestine (LI) of rats across different age groups from newborn to senescence using immunohistochemistry. In the MP of the SI and LI, the percentage of SST-IR neurons significantly increased during early postnatal development from 12 ± 2.4 (SI) and 13 ± 3.0 (LI) in newborn rats to 23 ± 1.5 (SI) and 18 ± 1.6 (LI) in 20-day-old animals, remaining stable until 60 days of age. The proportion of SST-IR cells then decreased in aged 2-year-old animals to 14 ± 2.0 (SI) and 10 ± 2.6 (LI). In the SP, the percentage of SST-IR neurons significantly rose from 22 ± 3.2 (SI) and 23 ± 1.7 (LI) in newborn rats to 42 ± 4.0 in 20-day-old animals (SI) and 32 ± 4.9 in 30-day-old animals (LI), before declining in aged 2-year-old animals to 21 ± 2.6 (SI) and 28 ± 7.4 (LI). Between birth and 60 days of age, 97-98% of SST-IR neurons in the MP and SP colocalized with ChAT in both plexuses of the SI and LI. The percentage of SST/ChAT neurons decreased in old rats to 85 ± 5.0 (SI) and 90 ± 3.8 (LI) in the MP and 89 ± 3.2 (SI) and 89 ± 1.6 (LI) in the SP. Conversely, in young rats, only a few SST-IR neurons colocalized with nNOS, but this percentage significantly increased in 2-year-old rats. The percentage of SST/NPY-IR neurons exhibited considerable variation throughout postnatal development, with no significant differences across different age groups in both the MP and SP of both intestines. No colocalization of SST with GFAP was observed in any of the studied animals. In conclusion, the expression of SST in enteric neurons increases in young rats and decreases in senescence, accompanied by changes in SST colocalization with ChAT and nNOS.

ReFerm®: a postbiotic fermented oat gruel composition is reducing mast cell degranulation in the colon of patients with irritable bowel syndrome.

Background: Irritable bowel syndrome (IBS) is a highly prevalent gastrointestinal disorder that affects ~4% of the global population. ReFerm® is a postbiotic product derived from oat gruel fermented with Lactobacillus plantarum 299v, and it has been shown to have beneficial effects on intestinal permeability in patients with IBS. In this study, we investigated the effects of ReFerm® on regulators of intestinal permeability, namely mast cells and enteric glial cells. Materials and methods: A total of 30 patients with moderate to severe IBS were treated with an enema containing ReFerm® or a placebo twice daily. The patients underwent sigmoidoscopy with biopsies obtained from the distal colon at baseline and after 14 days of treatment. These biopsies were processed in two ways: some were fixed, embedded in paraffin, sectioned, and stained for mast cells and enteric glial cells; others were cryopreserved, lysed, and subjected to Western blotting to analyze the same markers. Results: Treatment with ReFerm®, but not the placebo, significantly reduced mast cell tryptase protein levels in the biopsy lysates. Although the number of mast cells remained unchanged in colonic biopsies, ReFerm® treatment significantly reduced mast cell degranulation, a result not observed in the placebo group. Neither ReFerm® or placebo treatment had an impact on total protein levels or the number of enteric glial cells in the biopsies. Conclusion: ReFerm® treatment significantly reduced both total mast cell tryptase levels and the degranulation of mast cells in colonic biopsies from patients with IBS, suggesting a decrease in mast cell activity as a potential mechanism underlying the beneficial effects of ReFerm®. However, further research is required to assess the molecular mechanisms through which ReFerm® operates in the colons of patients with IBS. Clinical trial registration: https://clinicaltrials.gov, identifier: NCT05475314.

Neuronal distribution in colorectal cancer: associations with clinicopathological parameters and survival.

Over the last years, insights in the cancer neuroscience field increased rapidly and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n=490) and an in-cohort validation population (n=529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissue were stained for neuronal subtype markers and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF and PGP9.5 positive nerve fibers were found within the tumor stroma and were mostly characterized by the neuronal subtype markers vasoactive intestinal protein (VIP) and neuronal nitric oxide synthase (nNOS), suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (p=0.025), independent of other prognostic factors (HR=2.31; 95% CI 1.33-4.03; p=0.003), but these results were not observed in the in-cohort validation group. PGP9.5 on the other hand, was associated with a worse CRC-specific survival in the in-cohort validation (p=0.046) but not in the study population. This effect disappeared in multivariate analyses (HR=0.81; 95% CI 0.50-1.32; p=0.393) indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

Genomic stress in diseases stemming from defects in the second brain.

This review discusses the less-explored realm of DNA damage and repair within the enteric nervous system (ENS), often referred to as the "second brain." While the central nervous system has been extensively studied for its DNA repair mechanisms and associated neuropathologies, the ENS, which can autonomously coordinate gastrointestinal function, experiences unique challenges and vulnerabilities related to its genome integrity. The susceptibility of the ENS to DNA damage is exacerbated by its limited protective barriers, resulting in not only endogenous genotoxic exposures, such as oxidative stress, but also exogenous threats, such as ingested environmental contaminants, local inflammatory responses, and gut dysbiosis. Here, we discuss the evidence for DNA repair defects in enteric neuropathies, most notably, the reported relationship between inherited mutations in RAD21 and LIG3 with chronic intestinal pseudo-obstruction and mitochondrial gastrointestinal encephalomyopathy disorders, respectively. We also introduce the lesser-recognized gastrointestinal complications in DNA repair syndromes, including conditions like Cockayne syndrome. The review concludes by pointing out the potential role of DNA repair defects in not only congenital disorders but also aging-related gut dysfunction, as well as the crucial need for further research to establish direct causal links between DNA damage accumulation and ENS-specific pathologic phenotypes.

Dietary Exposure to Acrylamide Has Negative Effects on the Gastrointestinal Tract: A Review.

Changing eating habits and an increase in consumption of thermally processed products have increased the risk of the harmful impact of chemical substances in food on consumer health. A 2002 report by the Swedish National Food Administration and scientists at Stockholm University on the formation of acrylamide in food products during frying, baking and grilling contributed to an increase in scientific interest in the subject. Acrylamide is a product of Maillard's reaction, which is a non-enzymatic chemical reaction between reducing sugars and amino acids that takes place during thermal processing. The research conducted over the past 20 years has shown that consumption of acrylamide-containing products leads to disorders in human and animal organisms. The gastrointestinal tract is a complex regulatory system that determines the transport, grinding, and mixing of food, secretion of digestive juices, blood flow, growth and differentiation of tissues, and their protection. As the main route of acrylamide absorption from food, it is directly exposed to the harmful effects of acrylamide and its metabolite-glycidamide. Despite numerous studies on the effect of acrylamide on the digestive tract, no comprehensive analysis of the impact of this compound on the morphology, innervation, and secretory functions of the digestive system has been made so far. Acrylamide present in food products modifies the intestine morphology and the activity of intestinal enzymes, disrupts enteric nervous system function, affects the gut microbiome, and increases apoptosis, leading to gastrointestinal tract dysfunction. It has also been demonstrated that it interacts with other substances in food in the intestines, which increases its toxicity. This paper summarises the current knowledge of the impact of acrylamide on the gastrointestinal tract, including the enteric nervous system, and refers to strategies aimed at reducing its toxic effect.

Comparison of the Influence of Bisphenol A and Bisphenol S on the Enteric Nervous System of the Mouse Jejunum.

Bisphenols are dangerous endocrine disruptors that pollute the environment. Due to their chemical properties, they are globally used to produce plastics. Structural similarities to oestrogen allow bisphenols to bind to oestrogen receptors and affect internal body systems. Most commonly used in the plastic industry is bisphenol A (BPA), which also has negative effects on the nervous, immune, endocrine, and cardiovascular systems. A popular analogue of BPA-bisphenol S (BPS) also seems to have harmful effects similar to BPA on living organisms. Therefore, with the use of double immunofluorescence labelling, this study aimed to compare the effect of BPA and BPS on the enteric nervous system (ENS) in mouse jejunum. The study showed that both studied toxins impact the number of nerve cells immunoreactive to substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), the neuronal isoform of nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VAChT). The observed changes were similar in the case of both tested bisphenols. However, the influence of BPA showed stronger changes in neurochemical coding. The results also showed that long-term exposure to BPS significantly affects the ENS.

Enteric neuropathy in diabetes: Implications for gastrointestinal function.

Diabetes, commonly known for its metabolic effects, also critically affects the enteric nervous system (ENS), which is essential in regulating gastrointestinal (GI) motility, secretion, and absorption. The development of diabetes-induced enteric neuropathy can lead to various GI dysfunctions, such as gastroparesis and irregular bowel habits, primarily due to disruptions in the function of neuronal and glial cells within the ENS, as well as oxidative stress and inflammation. This editorial explores the pathophysiological mechanisms underlying the development of enteric neuropathy in diabetic patients. Additionally, it discusses the latest advances in diagnostic approaches, emphasizing the need for early detection and intervention to mitigate GI complications in diabetic individuals. The editorial also reviews current and emerging therapeutic strategies, focusing on pharmacological treatments, dietary management, and potential neuromodulatory interventions. Ultimately, this editorial highlights the necessity of a multidisciplinary approach in managing enteric neuropathy in diabetes, aiming to enhance patient quality of life and address a frequently overlooked complication of this widespread disease.

Autonomic and Enteric Profiling May Help Predict Response to Diverse Obesity Therapies.

PURPOSE: Changes in autonomic (ANS) and enteric nervous systems (ENS) may be involved in pathogenesis of obesity. We hypothesized that baseline autonomic and enteric parameters may predict outcomes of diverse obesity therapies. MATERIAL AND METHODS: We studied ANS and ENS physiology in 37 patients (8 male, 29 female, age 45 years, weight 129.7 kg) at 4 centers in patients undergoing medical (9: low-calorie diet) versus invasive (22: 16 sleeve, 6 bypass) and semi-invasive (6: 2 band, 2 high energy stimulation, 2 aspiration) weight loss therapies. Weight loss was reported as percent weight loss from baseline to latest values at 1 year and in some up to 5 years; classified as < or > /= 20% for each group. ANS testing included sympathetic adrenergic function by measuring reflex vasoconstriction and postural adjustment ratio. ENS was measured non-invasively using cutaneous low-resolution electrogastrogram. RESULTS: Percent weight loss was greater with the invasive (28.5%) than semi-invasive (9.1%) or non-invasive low-calorie diet (4.4%) (p < .001). Percent weight loss at 1 year (and up to 5 years) corresponded to the adrenergic measure of postural adjustment ratio (r = .42, p = .012), total pulse amplitude at rest (r = .56, p < .001), and electrogastrogram standing-to-rest difference (r = .33, p = .056). CONCLUSION: Baseline autonomic and enteric function measures correspond to percentage with loss in this pilot study using diverse weight loss methods. Autonomic and enteric profiling has potential clinical use for evaluation and treatment of obesity but needed larger controlled trials.

Mechanisms of enteric neuropathy in diverse contexts of gastrointestinal dysfunction.

The enteric nervous system (ENS) commands moment-to-moment gut functions through integrative neurocircuitry housed in the gut wall. The functional continuity of ENS networks is disrupted in enteric neuropathies and contributes to major disturbances in normal gut activities including abnormal gut motility, secretions, pain, immune dysregulation, and disrupted signaling along the gut-brain axis. The conditions under which enteric neuropathy occurs are diverse and the mechanistic underpinnings are incompletely understood. The purpose of this brief review is to summarize the current understanding of the cell types involved, the conditions in which neuropathy occurs, and the mechanisms implicated in enteric neuropathy such as oxidative stress, toll like receptor signaling, purines, and pre-programmed cell death.

Oral Exposure to Microplastics Affects the Neurochemical Plasticity of Reactive Neurons in the Porcine Jejunum.

Plastics are present in almost every aspect of our lives. Polyethylene terephthalate (PET) is commonly used in the food industry. Microparticles can contaminate food and drinks, posing a threat to consumers. The presented study aims to determine the effect of microparticles of PET on the population of neurons positive for selected neurotransmitters in the enteric nervous system of the jejunum and histological structure. An amount of 15 pigs were divided into three groups (control, receiving 0.1 g, and 1 g/day/animal orally). After 28 days, fragments of the jejunum were collected for immunofluorescence and histological examination. The obtained results show that histological changes (injury of the apical parts of the villi, accumulations of cellular debris and mucus, eosinophil infiltration, and hyperaemia) were more pronounced in pigs receiving a higher dose of microparticles. The effect on neuronal nitric oxide synthase-, and substance P-positive neurons, depends on the examined plexus and the dose of microparticles. An increase in the percentage of galanin-positive neurons and a decrease in cocaine and amphetamine-regulated transcript-, vesicular acetylcholine transporter-, and vasoactive intestinal peptide-positive neurons do not show such relationships. The present study shows that microparticles can potentially have neurotoxic and pro-inflammatory effects, but there is a need for further research to determine the mechanism of this process and possible further effects.

Mature enteric neurons have the capacity to reinnervate the intestine with glial cells as their guide.

Here, we establish that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs, as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within 2 weeks. These studies illustrate the structural and functional reinnervation capacity of post-mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories.

Gut-directed therapy in Parkinson's disease.

Parkinson's disease (PD) is a common and slow-progressing neurodegenerative disorder characterized by motor and non-motor symptoms, including gastrointestinal (GI) dysfunctions. Over the last years, the microbiota-gut-brain (MGB) axis is emerging as a bacterial-neuro-immune ascending pathway that contributes to the progression of PD. Indeed, PD patients are characterized by changes in gut microbiota composition, alterations of intestinal epithelial barrier (IEB) and enteric neurogenic/inflammatory responses that, besides determining intestinal disturbances, contribute to brain pathology. In this context, despite the causal relationship between gut dysbiosis, impaired MGB axis and PD remains to be elucidated, emerging evidence shows that MGB axis modulation can represent a suitable therapeutical strategy for the treatment of PD. This review provides an overview of the available knowledge about the beneficial effects of gut-directed therapies, including dietary interventions, prebiotics, probiotics, synbiotics and fecal microbiota transplantation (FMT), in both PD patients and animal models. In this context, particular attention has been devoted to the mechanisms by which the modulation of MGB axis could halt or slow down PD pathology and, most importantly, how these approaches can be included in the clinical practice.

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.

Tetrodotoxin-resistant mechanosensitivity and L-type calcium channel-mediated spontaneous calcium activity in enteric neurons.

Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.

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.

Enteric neuropathy and the vagus nerve: Therapeutic implications.

Enteric neuropathies are characterized by abnormalities of gut innervation, which includes the enteric nervous system, inducing severe gut dysmotility among other dysfunctions. Most of the gastrointestinal tract is innervated by the vagus nerve, the efferent branches of which have close interconnections with the enteric nervous system and whose afferents are distributed throughout the different layers of the digestive wall. The vagus nerve is a key element of the autonomic nervous system, involved in the stress response, at the interface of the microbiota-gut-brain axis, has anti-inflammatory and prokinetic properties, modulates intestinal permeability, and has a significant capacity of plasticity and regeneration. Targeting these properties of the vagus nerve, with vagus nerve stimulation (or non-stimulation/ pharmacological methods), could be of interest in the therapeutic management of enteric neuropathies.

Biphenotypic Cells and α-Synuclein Accumulation in Enteric Neurons of Leucine-Rich Repeat Kinase 2 Knockout Mice.

BACKGROUND: Leucine-rich repeat kinase 2 is a molecule that is responsible for familial Parkinson's disease. Our previous findings revealed that leucine-rich repeat kinase 2 is expressed in the enteric nervous system. However, which cells in the enteric nervous system express leucine-rich repeat kinase 2 and whether leucine-rich repeat kinase 2 is associated with the structure of the enteric nervous system remain unclear. The enteric nervous system is remarkable because some patients with Parkinson's disease experience gastrointestinal symptoms before developing motor symptoms. AIMS: We established a leucine-rich repeat kinase 2 reporter mouse model and performed immunostaining in leucine-rich repeat kinase 2 knockout mice. METHODS: Longitudinal muscle containing the myenteric plexus prepared from leucine-rich repeat kinase 2 reporter mice was analyzed by immunostaining using anti-green fluorescent protein (GFP) antibody. Immunostaining using several combinations of antibodies characterizing enteric neurons and glial cells was performed on intestinal preparations from leucine-rich repeat kinase 2 knockout mice. RESULTS: GFP expression in the reporter mice was predominantly in enteric glial cells rather than in enteric neurons. Immunostaining revealed that differences in the structure and proportion of major immunophenotypic cells were not apparent in the knockout mice. Interestingly, the number of biphenotypic cells expressing the neuronal and glial cell markers increased in the leucine-rich repeat kinase 2 knockout mice. Moreover, there was accumulation of α-synuclein in the knockout mice. CONCLUSIONS: Our present findings suggest that leucine-rich repeat kinase 2 is a newly recognized molecule that potentially regulates the integrity of enteric nervous system and enteric α-synuclein accumulation.