Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.

Locally transplanted enteric glia improve functional and structural recovery in a rat model of spinal cord injury

BACKGROUND： We have previously reported that adult enteric glia （EG） facilitate the growth of transected dorsal root axons into the uninjured spinal cord to form functional connections with their targets. OBJECTIVE： The present study investigated the effects of EG on spinal cord function, tissue injury, and axonal regeneration following transplantation into injured rat spinal cords, according to histological and functional outcomes. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at McMaster University, Canada from January 2006 to March 2008. MATERIALS： EG were isolated from rat intestine, METHODS： One week following spinal cord crush, female Wistar rats were injected with an EG suspension （2 μL, 1 × 10^5/μL, n = 10） or with the same volume of fresh culture medium alone （control animals, n = 11）. The third group did not receive any injection following laminectomy and served as the sham-operated controls （n = 5）. MAIN OUTCOME MEASURES： Behavior was tested prior to transplantation and weekly following transplantation, with nine behavioral examinations in total. Open field, hind limb placement response foot orientation response, and inclined plane test were utilized. Immediately following the final behavioral examination, spinal cord T9 to L1 segments were harvested for immunohistochemical and hematoxylin-eosin staining to determine astroglial scarring, axonal regeneration and spinal cord lesion size. RESULTS： Rats with EG transplantation exhibited significantly better locomotor function with reduced tissue damage, compared with the control rats. Cystic cavities were present 2 months after injury in spinal cords from both control groups. In contrast, rats injected with EG did not present with cystic lesions. In addition, the injury site consisted of cellular material and nerve fibers, and axonal regeneration was apparent, with dense labeling of neurofilament-positive axons within the injury site. Moreover, regenerating axons were intimately associated with transplanted EG. CONCLUSION： These data indicated that EG enhanced functional improvement, which was associated with reduced tissue damage and axonal regeneration following transplantation into injured spinal cords.

Inhibition and reversal of growth cone collapse in adult sensory neurons by enteric glia-induced neurotrophic factors

Aim:Previous studies show enteric glia(EG)-conditioned medium promotes neurite outgrowth in adult dorsal root ganglia(DRG)derived sensory neurons.This EG-conditioned medium contains various neurotrophic factors,including nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),glial cell line-derived neurotropic factor(GDNF),and neurotrophin-3(NT-3).This study attempts to determine the importance of these neurotrophic factors in enabling DRG-derived sensory neuron axons to overcome the inhibitory guidance cues released from the glial scar.Methods:A Semaphorin 3A(SEMA3A)growth cone collapse model was used on cultured rat DRG.Neutralizing antibodies to each neurotrophic growth factor in question(NGF,BDNF,GDNF and NT-3)were applied to the EG-conditioned medium to evaluate the factor’s individual importance in preventing growth cone collapse.Results:EG-conditioned medium inhibits and reverses growth cone collapse in adult DRG neurons when added either 1 h before or concurrently with SEMA3A.When administered 40 min after the initial SEMA3A-induced collapse,EG-conditioned medium was able to reverse the growth cone collapse.Individual inhibition of all the neurotrophic factors,except for BDNF in the co-treatment setting,resulted in increased growth cone collapse.Conclusion:NGF,BDNF,GDNF,and NT-3 are all variably involved in preventing or reversing SEMA3A-induced growth cone collapse in pre-,co-,and post-treatment time settings.However,no individual neurotrophic factors appear to be essential to promoting neurite outgrowth.

Immune regulation of the gut-brain axis and lung-brain axis involved in ischemic stroke

Local ischemia often causes a series of inflammatory reactions when both brain immune cells and the peripheral immune response are activated.In the human body,the gut and lung are regarded as the key reactional targets that are initiated by brain ischemic attacks.Mucosal microorganisms play an important role in immune regulation and metabolism and affect blood-brain barrier permeability.In addition to the relationship between peripheral organs and central areas and the intestine and lung also interact among each other.Here,we review the molecular and cellular immune mechanisms involved in the pathways of inflammation across the gut-brain axis and lung-brain axis.We found that abnormal intestinal flora,the intestinal microenvironment,lung infection,chronic diseases,and mechanical ventilation can worsen the outcome of ischemic stroke.This review also introduces the influence of the brain on the gut and lungs after stroke,highlighting the bidirectional feedback effect among the gut,lungs,and brain.

P2X7 receptor blockade decreases inflammation,apoptosis,and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice

Clostridioides difficile(C.difficile)is the most common pathogen causing health care-associated infections.C.difficile TcdA and TcdB have been shown to activate enteric neurons;however,what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown.AIM To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation,cell death,and the changes in the enteric nervous system in mice.METHODS Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA(50μg/Loop)for 4 h.To investigate the role of the P2X7 receptor,Brilliant Blue G(50 mg/kg,i.p.),which is a nonspecific P2X7 receptor antagonist,or A438079(0.7μg/mouse,i.p.),which is a competitive P2X7 receptor antagonist,were injected one hour prior to TcdA challenge.Ileal samples were collected to analyze the expression of the P2X7 receptor(by quantitative real-time polymerase chain reaction and immunohistochemistry),the population of myenteric enteric neurons(immunofluorescence),histological damage,intestinal inflammation,cell death(terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling),neuronal loss,and S100B synthesis(immunohistochemistry).RESULTS TcdA upregulated(P<0.05)the expression of the P2X7 receptor gene in the ileal tissues,increasing the level of this receptor in myenteric neurons compared to that in control mice.Comparison with the control mice indicated that TcdA promoted(P<0.05)the loss of myenteric calretinin+(Calr)and choline acetyltransferase+neurons and increased the number of nitrergic+and Calr+neurons expressing the P2X7 receptor.Blockade of the P2X7 receptor decreased TcdAinduced intestinal damage,cytokine release[interleukin(IL)-1β,IL-6,IL-8,and tumor necrosis factor-α],cell death,enteric neuron loss,and S100B synthesis in the mouse ileum.CONCLUSION Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor,which promoted enteric neuron loss,S100B synthesis,tissue damage,inflammation,and cell death in the mouse ileum.These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C.difficile infection.