The Underlying Mechanisms Of Hypersensitivity Of DRG Neurons Of Rats After Noxious Colorectal Distention And The Role Of Lactobacillus Reuteri

Visceral pain is an important phenomenon of some gastrointestinal(GI) diseases, but the molecular and cellular mechanisms are still not clear.The hypersensitivity of the nociceptive receptor,the primary sensory neuron and the central nervous system (CNS) are the main causes of visceral hyperalgesia.The GI tract has the potential to provide subconscious and conscious awareness of injury.The physiological or non-noxious stimuli are transmitted through vagal afferents with their cell bodies in nodose ganglion.But the pain is transmitted through the spinal afferents with their cell bodies in the dorsal root ganglia(DRG).DRG neurons are classified into three main groups according to their sizes: large-sized(39-50μm diameter),medium-sized(33-38μm diameter) and small-sized (19-27μm diameter) neuron.The spinal afferent Aδand C fibers,which transmit pain input to the dorsal corner in the spinal cord,are originated from medium-sized and small-sized neurons.So,the two kinds DRG neurons are the crucial "gateway" of visceral pain transmission.Voltage-gated sodium(Nav) channels play fundamental roles in the generation and conduction of neuronal action potentials.They are comprised ofα-subunit forming the pore,and auxiliaryβ-subunits.To date,nine kinds ofα-subunits have been cloned, and Nav were correspondingly classified into nine types,Nav1.1-Nav1.9.According to their sensitivity to tetrodotoxin(TTX),sodium channels have been classified to TTX-sensitive channels(TTX-S) and TTX-resistant channels(TTX-R).Nav1.1, Nav1.2,Nav1.3,Nav1.4,Nav1.6 and Nav1.7 are TTX-S channels and Nav1.5,Nav1.8 and Nav1.9 are TTX-R channels.Nav1.3,Nav1.7,Nav1.8 and Nav1.9 are thought to be involved in the modulation of the pain sensation by modulating the neurons excitability.But the detailed mechanisms are not clear.BDNF(brain-derived neurotrophic factor) is a kind of neurotrophic factor and is widely distributed in the peripheral and central nervous system.It is synthesized in the DRG,transported to the central terminals of the primary afferents,released into the spinal dorsal horn,and binds to the trkB receptors on second-order sensory neurons.It might play significant roles in synaptic plasticity,signal transmission,learning and memory.Nowadays,more and more studies found that BDNF also functions as a mediator of pain.Furthermore,BDNF itself could modulate the hypersensitivity of the DRG cells in a paracrine or autocrine manner.Probiotics,including Lactobacillus reuteri(L.reuteri) and Bifidobacterium,are health-promoting commensal bacteria that exert therapeutic or preventative roles to some gastrointestinal diseases.Beneficial effects of probiotics have been described in traveler's diarrhea,irritable bowel syndrome,and inflammatory bowel disease.In addition,probiotics can modulate postinflammatory gut hypersensitivity and alter neuropeptide expression in neurons.Based on the above backgrounds,we hypothesized that the change of the DRG neuron sensitivity was involved in the visceral hyperalgesia in some GI disease,and this hypersensitity of the DRG neurons might caused by the changes of the expression and electrical property of the sodium channels on the soma of the DRG neuron. BDNF and probiotics might modulate the sensation of pain by modulating the sensitivity of DRG neurons.In order to test this hypothesis,we use repeated noxious colorectal distention(CRD) to make the rat model of visceral hyperalgesia and monitor the sensitivity of the DRG neurons by whole cell recording. [Objective]1 To explore the effects of noxious CRD on the excitability of rat DRG neurons.2 To explore the effect of CRD on the expression and function of voltage-gated sodium channels.3 To explore the expression of BDNF in DRG following CRD and the effect of exogenous BDNF on the excitability of DRG neuron.4 To explore if L.reuteri could modulate the hypersensitivity of DRG neurons after CRD.[Methods and results]1 The noxious CRD model1.1 Male adult Sprague-Dawley rats were anesthetized with ketamine(75mg/kg, i.p.) and xylazine(10mg/kg,i.p.).A midline laparotomy was performed,and the descending colon was carefully exposed.1, 1-dioctadecyl-3-3-3-3-tetramethylindocarbocyanine(DiI) was injected into six to ten sites in the colon wall.Then,the abdomen was closed.1.2 Forteen days later,the rats were anesthetized again as above and treated with repeated distention for 1h(80 mm Hg,30s on,30 s off) by Barostat system.The vehicle control group was anesthetized but not treated with CRD.2 Measure of myeloperoxidase(MPO) activityThe rats were sacrificed and the MPO activity of the distal colon was measured. The MPO activities did not change following CRD.3 The BDNF mRNA levels in DRG(PCR)The total mRNA were extracted from DRG and the amount of mRNA in DRG was measured by RT-PCR.The BDNF mRNA in DRG were significantly increased from 3 h to 24 h following CRD.4 The BDNF protein levels in DRG(ELISA)The total protein were extracted from DRG and the amount of BDNF protein was mesured by ELISA.The expression of BDNF protein in DRG were significantly increased from 1h to 24h following CRD. 5 Cell dissociation and cultureAfter CRD,the rat was sacrificed immediately and the spinal colum was completely removed,transferred to a beaker containing ice-cold sterile Krebs solution. DRG were exposed by dissection from both sides and collected from L1-S5 spinal levels.After incubated 50min in collagenase typeâ… (1mg/ml) and trypsin(0.25%) at 37℃,the dissociated neurons were plated onto 35-mm-diameter Petri dishes previously coated with PLL.The neurons were then incubated overnight at 37℃with 5%CO₂ and 95%O₂.6 Electrophysiological recording6.1 Spontaneous discharge in DRG neuronsThe spontaneous discharge of the DRG DiI labled neurons was recorded in current clamp mode,I=0.The frequency of the spontaneous discharge in no CRD group were 7.3%(6/82),and that in the CRD group it was 9.4%(6/64).There were no significant difference betweent the two groups.6.2 Passive membrane and active membrane parameters in DRG neurons.With the amplifier in current clamp mode,action potential and passive membrane characteristics were measured.After CRD,the rheobase of the DRG neurons was significantly reduced and the number of APs discharged at 2×and 3×rheobase were increased compared with that in no CRD group.6.3 The effects of CRD on sodium current in DRG.Only DiI-labled DRG neurons were selected for this study.The patch pipettes were pulled with resistance 2-4 MΩ.After establishment of a whole cell recording,series resistance were compensated to 85-90%.The results showed that the activation and recovery currents of TTX-S and slow TTX-R channels in the CRD group have no difference from that in no CRD group.But theV_1/2 of inaction current shifted to the right.This implies that CRD change the inactivation kinemics of the sodium channel.6.4 The effects of exogenous BDNF on the excitability of DRG neurons in CRD and no CRD groupsExogenous BDNF reduced the AP_dV/dt and the amplitude of AP of the DRG neuron in no DRG group.But in CRD group,exogenous BDNF increased the rheobase, decreased AP_dV/dt and the number of APs discharged at 3×rheobase.So,it it clear that exogenous BDNF decreased the excitability of the DRG neurons in CRD group.6.5 The effect of L.reuteri ingestion on the excitability of DRG neurons in CRD and no CRD groupsIn the DRG neuron of the CRD group,9 days of L.reuteri ingestion increase the rheobase,decreased the AP_dV/dt and decreased the number of APs discharged at 2×rheobase intensity stimulation..[Conclusion]1 Repeated 80mmHg CRD stimulation in distal colon can cause increased excitability of DRG neurons.2 CRD might increase the excitability of the DRG neuron by changing inactivation kinemics of sodium channels.3 Endogenous BDNF might downregulate the hypersensitivity of DRG neurons following CRD.4 Ingestion of L.reuteri downregulated the sensitivity of the DRG neurons following CRD.It may exert therapeutic or preventative role to visceral pain.