The Mechanism Of Selective Activation Of Basal Forebrain Cholinergic Neurons Attenuates Polymicrobial Sepsis-Induced Inflammation

Part 1 The Effect of Selective Activation of Basal Forebrain Cholinergic Neurons on Regulating Polymicrobial Sepsis-Induced InflammationObjective:Cholinergic projection neurons in the basal forebrain(BF)originate the major cholinergic output in the CNS and have been widely studied in the sleep-wake transition and the control of cortical activation,plasticity,and cognition.However,it remains unknown whether BF cholinergic(ch-BF)neurons exert an anti-inflammatory effect on sepsis.Methods:We asked whether activating ch-BF neurons has an anti-inflammatory effect on sepsis using ChAT-Channelrhodopsin2(ChR2)-EYFP optogenetic and wild-type(WT)mice.The optical fiber was coupled to a 473 nm laser under driver control.Light pulse trains(30 ms pulses at 20 Hz for 15 s once per minute for 30 min every 1.5 hr)were controlled using digital commands and a Master-8 pulse stimulator.Serum and tissue supernatants were used to analyze the protein levels of TNF-a,interleukin(IL)-6,and IL-10 using enzyme linked immunosorbent assay(ELISA).Results:When ch-BF neurons were photostimulated,serum levels of TNF-a were significantly lower after 3(p<0.01)and 12(p<0.001)hours in ChAT-lit septic mice than in ChAT-unlit septic mice.Serum levels of IL-6 were lower after 3 hours(p<0.05)and further lowered after 12 hours(p<0.001)in ChAT-lit mice.Our analysis of cytokine concentrations in spleen protein extracts produced similar results.Additionally,we found that TNF-a and IL-6 levels were lower at 3 and 12 hours after ch-BF neurons were photoactivated,whereas IL-10 levels were not significantly different between ChAT-lit and ChAT-unlit mice by CLP.There was no significant difference in the concentrations of TNF-a,IL-6,and IL-10 between WT and ChAT septic mice that were not photostimulated.However,there was no significant difference in survival between ChATlit septic mice and ChAT-unlit septic mice.Conclusions:These results indicate that photostimulating ch-BF neurons significantly alleviated systemic inflammatory responses,and especially the release of pro-inflammatory cytokines,following CLP surgery.Part 2 The Role of the Vagus Nerve on Systemic Inflammatory Response in Sepsis Induced by Photostimulating ch-BF NeuronsObjective:The efferent vagus nerve,through releasing acetylcholine,dominates systemic inflammation and inhibits the release of proinflammatory cytokines,depending on the a7-nicotinic acetylcholine receptor.This network is therefore called the"cholinergic anti-inflammatory pathway".To determine whether the vagus nerve is essential for the immunomodulatory function of ch-BF neurons during sepsis,we performed left cervical vagotomy before CLP surgery in photostimulated WT and ChAT mice.Methods:A ventral cervical midline incision was used to expose the left cervical vagus trunk,which was ligated using 4-0 silk sutures and excised at least 1 cm.The skin was then closed.In sham-operated mice,the left vagus nerve was exposed and isolated from the surrounding tissue but not transected.All animals were vagotomized 3 days before CLP.Serum and tissue supernatants were used to analyze the protein levels of TNF-a,and IL-6 using ELISA.Results:We observed that serum concentrations of TNF-a(p<0.01)and IL-6(p<0.05)were lower in ChAT septic mice that did not undergo left cervical vagotomy,and this effect was nearly abolished after 12 hours in ChAT septic mice that underwent left cervical vagotomy.Moreover,in ChAT mice,left cervical vagotomy restored IL-6 levels in spleen after 12 hours(p<0.05).Conclusions:These findings indicate that the vagus nerve is required for ch-BF neuronal photoactivation to modulate the systemic inflammatory response.Part 3 The Central Neuronal Circuits of Stimulating ch-BF Neurons Mediated the Anti-inflammation in SepsisObjective:To clarify the functional connection between ch-BF neurons and the peripheral immune response,we investigated neuronal activity in the nucleus of the vagus nerve after photoactivation of ChR2-expressing ch-BF neurons.Methods:We used c-Fos expression to screen photoactivation-induced neuronal activity in the whole brain.Results:We found that a large number of neurons in the BF region of photoactivated ChAT mice were c-Fos positive,and some of these c-Fos-positive neurons were also ChAT-positive,indicating that cholinergic neurons were activated by in vivo photostimulation.Interestingly,photoactivating ch-BF neurons also significantly activated c-Fos-positive neurons in the DMN/SolV.However,the number of c-Fos-positive neurons was significantly lower in the BF and DMN/SolV in ChAT mice not treated with photostimulation.Our analysis of the number of c-Fos-positive cells indicated that photostimulating ch-BF neurons in ChAT mice induced significantly more c-Fos expression in both the BF(p<0.01)and the DMN/SolV(p<0.001)than was observed in nonphotostimulated ChAT mice.Conclusions:These results collectively indicate that the neuronal connections between the BF and the DMN/SolV are involved in modulating the inflammatory response in sepsis.Part 4 The Mechanism of Dopaminergic Neurons in the the Dorsal Motor Nucleus of the Vagus(DMN)/Ventral Part of the Solitary Nucleus(SolV)Mediated the Cholinergic Anti-Inflammatory Effect of Vagus NerveObjective:To determine what types of neurons activated by photostimulation of ch-BF neurons,we examined the expression of tyrosine hydroxylase(TH),ChAT,and c-Fos in the DMN/SolV.Methods:Chemical splenic denervation was performed by injecting 6-OHDA(60-120?g)or saline into exteriorized spleens.The mice were then allowed to recover for 1 week before photostimulation.Results:We observed very few ChAT+c-Fos+ cells but a significant increase in the density of TH+c-Fos+cells in the DMN/SolV of the ChAT-lit mice(Th+c-Fos+cells vs ChAT+c-Fos+cells:35.2%4.6%vs 6.1%± 3.9%).Our results reveal that dopaminergic neurons,specifically labeled with TH5 composed the main type of neurons in the DMN/SolV,which are activated by photostimulation of ch-BF neurons.Nerve fibers in the spleen that originate in the celiac ganglion use norepinephrine as their primary neurotransmitter.We next sought to determine whether the dopaminergic neurons in the DMN/SolV mediated such an anti-inflammatory effect by locally injecting 6-OHDA to destroy dopaminergic neurons in the spleen.Surprisingly,destroying dopaminergic transmission to the spleen almost completely reversed the reduction in serum and spleen concentrations of TNF-a and IL-6 that was induced in photoactivated ChAT mice.Conclusions:Collectively,our data reveal that dopaminergic neurons in the DMN/SolV indeed mediated the modulation of the systemic inflammatory response of the vagus nerve.