Search Results (66)

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications. Full article

Chronic pain is a widespread global health problem with profound socioeconomic implications, affecting millions of people of all ages. Glial cells (GCs) in pain pathways play essential roles in the processing of pain signals. Dysregulation of GC activity contributes to chronic pain states, making them targets for therapeutic interventions. Non-pharmacological approaches, such as exercise, are strongly recommended for effective pain management. This review examines the link between exercise, regular physical activity (PA), and glial cell-mediated pain processing, highlighting its potential as a strategy for managing chronic pain. Exercise not only improves overall health and quality of life but also influences the function of GCs. Recent research highlights the ability of exercise to mitigate neuroinflammatory responses and modulate the activity of GCs by reducing the activation of microglia and astrocytes, as well as modulating the expression biomarkers, thereby attenuating pain hypersensitivity. Here, we summarize new insights into the role of exercise as a non-pharmacological intervention for the relief of chronic pain. Full article

The trigeminal nerve is the primary gateway through which the rabies virus enters the brain. Viral infection-related trigeminal neuritis is associated with certain clinical signs. This study investigated trigeminal ganglion histopathology in 92 rabid dogs. Trigeminal ganglionitis was classified into three pathological grades: mild, moderate, and severe. Immunostaining of selected sections was performed using antibodies against lymphocytes (CD3, CD20), stellate cells (glial fibrillary acidic protein, GFAP), macrophages (Iba-1, HLA-DR), ganglion cells (neurofilament, NF), and Schwann cells (S-100) to identify lesion cell types. In moderate and severe cases, double-immunofluorescence staining was performed to determine neuronophagia and Nageotte nodule cell types. Mild (13.0%) cases had minimal morphological changes in ganglion cells; moderate (56.5%) and severe (30.4%) cases showed infected ganglion cells and axons with degenerative necrosis, which were replaced by inflammatory cells. Immunohistochemically, viral antigens were detected in most ganglion cells in mild cases and were significantly reduced in severe cases. The number of CD3-, CD20-, GFAP-, and Iba-1-positive cells increased as the severity progressed, and neuronophagia and Nageotte nodules primarily comprised HLA-DR-positive cells. These findings suggest that the rabies virus reaches the trigeminal ganglion via ascending or descending routes and induces trigeminal neuropathological changes, contributing to neurological symptoms in rabid dogs. Full article

The beta-3 adrenergic receptor (β3-AR), whose expression is modulated by oxygen levels, was found to play a key role in organ maturation, and its agonism was reported to mitigate hyperoxia-induced large bowel damage by preventing organ hypoplasia, preserving epithelial integrity, vascularization, and the neurochemical coding in the colonic myenteric plexus. This study explored the effects of β3-AR agonism in preventing hyperoxia-related alterations on the ileal enteric nervous system (ENS). Sprague–Dawley rat pups were reared under normoxia or hyperoxia (85%) during the first two weeks after birth and treated or not with the β3-AR agonist BRL37344 at 1, 3, or 6 mg/kg. Hyperoxia caused an imbalance of inhibitory nitrergic and excitatory cholinergic neurons in both the myenteric and submucosal plexuses and decreased the amounts of neurons in the submucosal plexus and that of S100β⁺ and GFAP⁺ glial cells in the myenteric plexus. Administration of 3 mg/kg BRL37344 preserved the neuronal chemical coding and partially prevented the loss of myenteric GFAP⁺ glial cells, while it did not counteract submucosal neuronal loss. Our findings indicate the potential of β3-AR agonism as a new therapeutic strategy for hyperoxia-induced ileal ENS alterations. Full article

Chronic pain is a global health problem with major socioeconomic implications. Drug therapy for chronic pain is limited, prompting search for non-pharmacological treatments. One such approach is physical exercise, which has been found to be beneficial for numerous health issues. Research in recent years has yielded considerable evidence for the analgesic actions of exercise in humans and experimental animals, but the underlying mechanisms are far from clear. It was proposed that exercise influences the pain pathways by interacting with the immune system, mainly by reducing inflammatory responses, but the release of endogenous analgesic mediators is another possibility. Exercise acts on neurons and glial cells in both the central and peripheral nervous systems. This review focuses on the periphery, with emphasis on possible glia–neuron interactions. Key topics include interactions of Schwann cells with axons (myelinated and unmyelinated), satellite glial cells in sensory ganglia, enteric glial cells, and the sympathetic nervous system. An attempt is made to highlight several neurological diseases that are associated with pain and the roles that glial cells may play in exercise-induced pain alleviation. Among the diseases are fibromyalgia and Charcot–Marie–Tooth disease. The hypothesis that active skeletal muscles exert their effects on the nervous system by releasing myokines is discussed. Full article

The blood–brain barrier and glial cells, particularly astrocytes, interact with each other in neuroimmune diseases. In the inflammatory environment typical of these diseases, alterations in vascular endothelial cell surface molecules and weakened cell connections allow immune cells and autoantibodies to enter the central nervous system. Glial cells influence the adhesion of endothelial cells by changing their morphology and releasing various signaling molecules. Multiple sclerosis has been the most studied disease in relation to vascular endothelial and glial cell interactions, but these cells also significantly affect the onset and severity of other neuroimmune conditions, including demyelinating and inflammatory diseases. In this context, we present an overview of these interactions and highlight how they vary across different neuroimmune diseases. Full article

Diabetes mellitus is associated with changes in intestinal morphology and the enteric nervous system. We previously reported constipation in Goto-Kakizaki (GK) rats, a non-obese model for type 2 diabetes mellitus. Aim: The morpho-quantitative analysis of myenteric plexus neurons in the small and large intestines of 120-day-old male GK rats was investigated. Methods: The diabetes was confirmed by high fasting blood glucose levels. The myenteric plexus was evaluated through wholemount immunofluorescence. The morpho-quantitative analyses included evaluating neuronal density (neurons per ganglion) of the total neuronal population, the cholinergic and nitrergic subpopulations, and enteric glial cells per ganglion. The cell body area of 100 neurons per segment per animal was measured. Results: The total neurons and nitrergic subpopulation were unaltered in the GK rats’ small and large intestines. The cholinergic subpopulation exhibited decreased density in the three segments of the small intestine and an increased number in the proximal colon of the GK rats. The number of enteric glial cells increased in the ileum of the GK rats, which could indicate enteric gliosis caused by the intestinal inflammatory state. The area of the cell body was increased in the total neuronal population of the jejunum and ileum of the GK rats. Frequency histograms of the cell body area distribution revealed the contribution of cholinergic neurons to larger areas in the jejunum and nitrergic neurons in the ileum. Conclusion: The constipation previously reported in GK rats might be explained by the decrease in the density of cholinergic neurons in the small intestine of this animal model. Full article

The enteric nervous system (ENS) is a fundamental component of the gastrointestinal system, composed of a vast network of neurons and glial cells. It operates autonomously but is interconnected with the central nervous system (CNS) through the vagus nerve. This communication, known as the gut–brain axis, influences the bidirectional communication between the brain and the gut. Background/Objectives: This study aimed to review neurological pathologies related to the ENS. Methods: To this end, a comprehensive literature search was conducted in the “PubMed” database. Articles available in “free format” were selected, applying the filters “Humans” and limiting the search to publications from the last ten years. Results: The ENS has been linked to various neurological diseases, from autism spectrum disorder to Parkinson’s disease including neurological infection with the varicella zoster virus (VZV), even sharing pathologies with the CNS. This finding suggests that the ENS could serve as an early diagnostic marker or therapeutic target for neurological diseases. Gastrointestinal symptoms often precede CNS symptoms, and the ENS’s accessibility aids in diagnosis and treatment. Parkinson’s patients may show intestinal lesions up to twenty years before CNS symptoms, underscoring the potential for early diagnosis. However, challenges include developing standardized diagnostic protocols and the uneven distribution of dopaminergic neurons in the ENS. Continued research is needed to explore the ENS’s potential in improving disease prognosis. Conclusions: The ENS is a promising area for early diagnosis and therapeutic development. Nevertheless, it is essential to continue research in this area, especially to gain a deeper understanding of its organization, function, and regenerative capacity. Full article

Gastrointestinal (GI) toxicity is a common side effect in patients undergoing oxaliplatin (OxPt)-based chemotherapy for colorectal cancer (CRC). Frequently, this complication persists in the long term and could affect the efficacy of the treatment and the patient’s life quality. This long-term GI toxicity is thought to be related to OxPt-induced enteral neuropathy. AmotL2 is a member of the Angiomotin family of proteins, which play a role in cell survival, neurite outgrowth, synaptic maturation, oxidative stress protection, and inflammation. In order to assess the role of AmotL2 in OxPt-induced enteral neuropathy, we studied the expression of AmotL2 in cells of the enteric nervous system (ENS) of untreated and OxPt-treated CRC patients and its relationship with inflammation, using immunofluorescence confocal microscopy. Our results in human samples show that the total number of neurons and glial cells decreased in OxPt-treated patients, and TNF-α and AmotL2 expression was increased and colocalized in both neurons and glia. AmotL2 differential expression between OxPt-treated and untreated CRC patients shows the involvement of this scaffold protein in the inflammatory component and toxicity by OxPt in the ENS. Full article

Clostridioides difficile (C. difficile) is responsible for a spectrum of nosocomial/antibiotic-associated gastrointestinal diseases that are increasing in global incidence and mortality rates. The C. difficile pathogenesis is due to toxin A and B (TcdA/TcdB), both causing cytopathic and cytotoxic effects and inflammation. Recently, we demonstrated that TcdB induces cytopathic and cytotoxic (apoptosis and necrosis) effects in enteric glial cells (EGCs) in a dose/time-dependent manner and described the underlying signaling. Despite the role played by lipids in host processes activated by pathogens, to counter infection and/or induce cell death, to date no studies have investigated lipid changes induced by TcdB/TcdA. Here, we evaluated the modification of lipid composition in our in vitro model of TcdB infection. Apoptosis, cell cycle, cell viability, and lipidomic profiles were evaluated in EGCs treated for 24 h with two concentrations of TcdB (0.1 ng/mL; 10 ng/mL). In EGCs treated with the highest concentration of TcdB, not only an increased content of total lipids was observed, but also lipidome changes, allowing the separation of TcdB-treated cells and controls into different clusters. The statistical analyses also allowed us to ascertain which lipid classes and lipid molecular species determine the clusterization. Changes in lipid species containing inositol as polar head and plasmalogen phosphatidylethanolamine emerged as key indicators of altered lipid metabolism in TcdB-treated EGCs. These results not only provide a picture of the phospholipid profile changes but also give information regarding the lipid metabolism pathways altered by TcdB, and this might represent an important step for developing strategies against C. difficile infection. Full article

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus—both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field. Full article

The enteric nervous system (ENS), consisting of neurons and glial cells, is situated along the gastrointestinal (GI) tract’s wall and plays a crucial role in coordinating digestive processes. Recent research suggests that the optimal functioning of the GI system relies on intricate connections between the ENS, the intestinal epithelium, the immune system, the intestinal microbiome, and the central nervous system (CNS). Inflammatory bowel disease (IBD) encompasses a group of chronic inflammatory disorders, such as Crohn’s disease (CD) and ulcerative colitis (UC), characterized by recurring inflammation and damage to the GI tract. This review explores emerging research in the dynamic field of IBD and sheds light on the potential role of ENS alterations in both the etiology and management of IBD. Specifically, we delve into IBD-induced enteric glial cell (EGC) activation and its implications for persistent enteric gliosis, elucidating how this activation disrupts GI function through alterations in the gut–brain axis (GBA). Additionally, we examine IBD-associated ENS alterations, focusing on EGC senescence and the acquisition of the senescence-associated secretory phenotype (SASP). We highlight the pivotal role of these changes in persistent GI inflammation and the recurrence of IBD. Finally, we discuss potential therapeutic interventions involving senotherapeutic agents, providing insights into potential avenues for managing IBD by targeting ENS-related mechanisms. This approach might represent a potential alternative to managing IBD and advance treatment of this multifaceted disease. Full article

Cortical traumatic brain injury (TBI) is a major cause of cognitive impairment accompanied by motor and behavioral deficits, and there is no effective treatment strategy in the clinic. Cell transplantation is a promising therapeutic strategy, and it is necessary to verify the survival and differentiation of cells after transplantation in large animal models like rhesus monkeys. In this study, we transplanted neural stem cells (NSCs) and simultaneously injected basic fibroblast growth factor/epidermal growth factor (bFGF/EGF) into the cortex (visual and sensory cortices) of rhesus monkeys with superficial TBI. The results showed that the transplanted NSCs did not enter the cerebrospinal fluid (CSF) and were confined to the transplantation site for at least one year. The transplanted NSCs differentiated into mature neurons that formed synaptic connections with host neurons, but glial scar formation between the graft and the host tissue did not occur. This study is the first to explore the repairing effect of transplanting NSCs into the superficial cerebral cortex of rhesus monkeys after TBI, and the results show the ability of NSCs to survive long-term and differentiate into neurons, demonstrating the potential of NSC transplantation for cortical TBI. Full article

The brain–gut axis has been identified as an important contributor to the physiopathology of Parkinson’s disease. In this pathology, inflammation is thought to be driven by the damage caused by aggregation of α-synuclein in the brain. Interestingly, the Braak’s theory proposes that α-synuclein misfolding may originate in the gut and spread in a “prion-like” manner through the vagus nerve into the central nervous system. In the enteric nervous system, enteric glial cells are the most abundant cellular component. Several studies have evaluated their role in Parkinson’s disease. Using samples obtained from patients, cell cultures, or animal models, the studies with specific antibodies to label enteric glial cells (GFAP, Sox-10, and S100β) seem to indicate that activation and reactive gliosis are associated to the neurodegeneration produced by Parkinson’s disease in the enteric nervous system. Of interest, Toll-like receptors, which are expressed on enteric glial cells, participate in the triggering of immune/inflammatory responses, in the maintenance of intestinal barrier integrity and in the configuration of gut microbiota; thus, these receptors might contribute to Parkinson’s disease. External factors like stress also seem to be relevant in its pathogenesis. Some authors have studied ways to reverse changes in EGCs with interventions such as administration of Tryptophan-2,3-dioxygenase inhibitors, nutraceuticals, or physical exercise. Some researchers point out that beyond being activated during the disease, enteric glial cells may contribute to the development of synucleinopathies. Thus, it is still necessary to further study these cells and their role in Parkinson’s disease. Full article

Recent discoveries have shown that enteric glial cells play an important role in different neurodegenerative disorders, such as Parkinson’s disease (PD), which is characterized by motor dysfunctions caused by the progressive loss of dopaminergic neurons in the substance nigra pars compacta and non-motor symptoms including gastrointestinal dysfunction. In this study, we investigated the modulatory effects of the flavonoid rutin on the behavior and myenteric plexuses in a PD animal model and the response of enteric glia. Adult male Wistar rats were submitted to stereotaxic injection with 6-hydroxydopamine or saline, and they were untreated or treated with rutin (10 mg/kg) for 14 days. The ileum was collected to analyze tissue reactivity and immunohistochemistry for neurons (HuC/HuD) and enteric glial cells (S100β) in the myenteric plexuses. Behavioral tests demonstrated that treatment with rutin improved the motor capacity of parkinsonian animals and improved intestinal transit without interfering with the cell population; rutin treatment modulated the reactivity of the ileal musculature through muscarinic activation, reducing relaxation through the signaling pathway of nitric oxide donors, and increased the longitudinal contractility of the colon musculature in parkinsonian animals. Rutin revealed modulatory activities on the myenteric plexus, bringing relevant answers regarding the effect of the flavonoid in this system and the potential application of PD adjuvant treatment. Full article