Properties And Mechanisms Of Conduction Failure In Polymodal C Fibers And Its Role In Regulating Chronic Pain

The unmyelinated fiber is the peripheral axon of the small-sized dorsal root ganglionneuron (DRG), and it transmits nociceptive information from peripheral nerve terminals tothe DRG neurons. The axon is regard as a relatively simple ralay line, and it usuallycarries signals over long distances faithfully. However, more and more theoretical andexperimental evidence showed obvious slowing of conduction velocity (CVs) andconduction failure in unmyelinated axons, and changing of the pattern of spikes and thenumber of spikes. Those phenomena indicate that action potentials may not be alwaystransmitted faithfully along the unmyelinated axon, but can be processed and integratedduring signal transmittion.In our previous work, we observed that unmyelinated C fibers have conduction failue.Three types of conduction failures were identified, no conduction failure, regularconduction failure and irregular conduction failure. My previous work shows that thedegree of conduction failure was significantly decreased in polymodal C fibers ofcoccygeal nerve from STZ-induced diabetic rats, and accompaniying by increased initial conduction velocity (CVi) and decreased slowing of conduction velocity (CVs). Whatmechanism of conduction failure is in the axon of C fiber? And what is the significance ofreduced conduction failure in pathological pain conditions? These questions will beaddressed in this study.This study aimed to clarify the properties of conduction failure using single fiberrecording in the cccygeal nerve from CFA rats in vivo. The characteristics of conductionfailure in polymodal C fibers induced by electrical stimulation with different frequencywas studied and analyzed. We also study the ionic mechanism underlying the conductionfailure in small-sized dorsal root ganglion (DRG) neurons using patch clamp. And then westudy the expression of HCN1and HCN2in small–sized DRG neurons and C fibers usingimmunofluorescent staining and western blot. At last, we study the effects of ZD7288onmechanical allodynia and thermal hyperalgesia by perineural injection of differentconcentrations of ZD7288.The results include four parts as follows:Part1: Properties of conduction failureThe properties of conduction failure were studied in polymodal C fibers of coccygealnerves from rats3to5days after treatment with saline or CFA. Action potentials wereevoked by electrical stimulation with different frequency at the receptive field. Drugs wereapplicated at the nerve trunk between the stimulating electrode and the recordingelectrode.1. We classified C fibers as polymodal C fibers and mechanical sensitive C fibers bystimulating the receptive field with nociceptive mechanical and thermal stimulation. Cfibers only responsed to mechanical stimulation was defined as mechanial sensitive Cfibers, and responsed to both stimulations was defined as polymodal C fibers. In this study,we found that the degree of conduction failure in polymodal C fibers was significantlylarger than that from mechanical sensitive C fibers (P <0.05). The degree of conductionfailure of polymodal C fibers by electrical stimulation with10Hz was45.8%±4.7%, andwhich in mechanical C fibers was5.8%±2.4%. Compared with mechanical sensitive Cfibers, the degree of slowing of conduction velocity (CVs) was markedly decreased in polymodal C fibers (P <0.05), the CVs in polymodal C fibers was35.5%±1.5%underelectrical stimulation of5Hz, and the CVs inmechanical C fibers was35.5%±1.5%.However, the initial conduction velocity (CVi) had no significant differnce (0.75±0.04m/s versus0.82±0.07m/s).2. The degree of conduction failure increased in a frequency-dependent (2,5and10Hz)manner, and it was significantly reduced in polymodal C fibers from CFA rats comparedwith control rats (P <0.05). The degree of conduction failure in control group underdifferent frequency electrcal stimulation (2,5and10Hz) was5.36%±2.51%,30.99%±4.08%and50.55%±4.39%, respectively. It was0.73%±0.31%,8.03%±1.83%and16.58%±3.46%in CFA group. We also found that the intial of conduction velocity (CVi)was significantly increased in polymodal C fibers from CFA rats than that from controlrats under electrical stimulation of2Hz (P <0.05), the CVi in control group was0.69±0.02m/s, and in CFA group was0.79±0.02m/s. But the degree of slowing of conductionvelocity (CVs) was dramaticlly reduced compared with control rats (P <0.05), the CVs incontrol group was24.96%±0.7%, and it was19.51%±1.1%in CFA group.3. We found that ZD7288(5,10,20and40μM), a specific blocker of Ih current,enhanced the degree of conduction failure under electrical stimulation with10Hz in adose-dependent manner (P <0.05). Under electrical stimulation with10Hz, the degree ofconduction failure before ZD7288was15.44%±2.61%, and after application ZD7288, itwas30.63%±2.56%(5μM),32.51%±1.82%(10μM),39.35%±3.25%(20μM) and50.93%±3.64%(40μM). The interspike interval (ISI) was increased by application ofZD7288. We also observed that ZD7288(40μM) significantly enhanced the slowing ofconduction velocity (P <0.05), the CVs before ZD7288was19.11%±1.41%, and turnedto22.51%±2.42%after application of ZD7288(40μM). But had no obvious effects onthe initial conduction velocity (0.78±0.02m/s versus0.75±0.02m/s).4. ZD7288(40μM) increased the degree of conduction failure in an activity-dependentmanner under different electrical stimulation (2,5and10Hz, P <0.05). Beforeapplication of ZD7288, the degree of conduction failure was0(2Hz),3.73%±2.14%(5Hz),15.44%±2.6%(10Hz). After application of ZD7288, the degree of conduction failure was2.08%±2.08%(2Hz),15.67%±4.21%(5Hz),50.93%±3.64%(10Hz).5. ZD7288selectively blocked the conduction of polymodal C fibers but not A fibersunder the same frequency electrical stimulation (10Hz).6. There was about20%C fibers had spontaneous discharges3-5days after injection ofCFA, and the frequency of spontaneous firings were significantly inhibited in adose-dependent manner by application of differnet ZD7288(40μM，150μM and500μM)on the nerve trunk between receptive field and recording elctrode (P <0.05). The firingfrequency was1.32±0.18Hz before application of ZD7288, and turned to be0.90±0.11Hz,0.59±0.05Hz and0.45±0.03Hz after application of ZD7288(40μM，150μM，500μM) respectivly. The interspike interval was also change by ZD7288.7. α-DTX (0.5nM), a selective blocker of low threshold potassium current, significantlyreduced the degree of conduction failure when application on the nerve trunk betweenstimulating electrode and recording electrode (P <0.05). The degree of conduction failurewas35.45%±4.23%before application of α-DTX, but decreased to8.42%±1.43%afterapplication of α-DTX (0.5nM). The initial conduction velocity was significantly increasedafter treatment of α-DTX (0.5nM, P <0.05). The initial conduction velocity was0.69±0.02m/s before application of α-DTX, and turned to be0.78±0.01m/s after treatment ofα-DTX. The degree of slowing of conduction velocity was significantly reduced (P <0.05). The CVs was23.5%±0.71%before treatment of α-DTX, and turned to be17.1%±0.79%after application of α-DTX.Part2: Ionic mechanisms underlying the conduction failureThe diameter of polymodal C fiber was too thin to record by patch clamp, and it was hardto directly study the properties of ionic channels on axons. The unmyelinated C fiber wasthe peripheral part of DRG neuron, and proteins on it were synthesized in the soma andthen transported from the soma to the axon. So we study the electrophysiologicalproperties of small-sized DRG neuron to indirectly reflect the alteration in unmyelinated Cfibers.1. The active membrane properties of small-sized DRG neurons from CFA rats weresignificantly different from control rats. The amplitude and max-rise slope were significantly increased in DRG neurons from CFA rats compared with control rats. But thehalf-width and threshold of acton potential were remarkedly decreased in small-sizedDRG neurons. These data indicated that the excitability of small-sized DRG neurons wassignificantly increased after injectionof CFA. More importantly, compared with contolneurons, neurons from CFA rats evoked more numbers of action potentials responded tostep pulses (500ms). But there was no significant difference of resting membrane betweentwo groups.2. When application of α-DTX (100nM) in the recording chamber5minutes, we foundthat the number of action potentials evoked by step stimulus were significantly increased.That means α-DTX can increase the excitability of small-sized DRG neurons.3. We then examine the alternations of α-DTX sensitive potassium current, and wefound that α-DTX sensitive potassium current was markedly reduced in small-sized DRGneurons from CFA rats compared with control rats (P <0.05). However, the activityproperty had no significant difference between two groups.4. We also found that Ih current was significantly increased in small-sized DRG neuronsafter CFA (P <0.05).5. Both the amplitude and half-width of after hyperpolarization potential of spikes insmall-sized DRG neurons from CFA rats was significantly decreased compared with thatfrom control rats (P <0.05). The amplitude and half-width of AHP in control group were12.2±0.62mV and30.2±2.5ms, respectively. The amplitude and half-width of AHP inCFA group were8.1±0.74mV and15.8±2.7ms, respectively. When10minutes afterapplication ZD7288(40μM) in the recording chamber, both the amplitude and half-widthwere remarkedly increased in small-sized DRG neurons from CFA rats (P <0.05). Theamplitude and half-width of AHP in CFA group with ZD7288were10.5±0.53mV and25.6±3.3ms, respectively.Part3: Ionic mechanisms underlying the conduction failure In this section, we study the expression of HCN1and HCN2in the small-sized DRGneurons and C fibers from CFA rats and control rats.1. The expression of HCN1in both DRG neurons and C fibers was significantlyincreased from CFA rats compared with control rats (P <0.05). CFA rats showed muchstronger HCN1immunoflurescence intensity in peripherin-positive DRG neurons and Cfibers than that from control rats. The immunoflurescence intensity of HCN1in small DRGneurons and C fibers from control rats were23.4±3.8and17.6±3.5, respectively. Theimmunoflurescence intensity of HCN1in small DRG neurons and C fibers from CFA ratswere49.5±4.3and45.8±5.4, respectively.2. The expression of HCN2in both DRG neurons and C fibers was significantlyincreased from CFA rats compared with control rats (P <0.05). CFA rats showed muchstronger HCN2immunoflurescence intensity in peripherin–positive DRG neurons and Cfibers than that from control rats. The immunoflurescence intensity of HCN2in small DRGneurons and C fibers from control rats were26.5±6.8and20.3±4.2, respectively. Theimmunoflurescence intensity of HCN2in small DRG neurons and C fibers from CFA ratswere64.5±8.6and44.6±7.6, respectively.Part4: ZD7288partly inhibited mechanical allodynia and thermal hyperalgesia inCFA ratsIn this part we studied the effects of ZD7288on mechanical allodynia and thermalhyperalgesia by perineural injection, and we aslo studied the effects of ZD7288on motorfunction and heart rate.1. We found that ZD7288partly inhibited mechanical allodynia and thermalhyperalgesia in a dose-dependent and time-dependent manner by perineural injection.2. ZD7288had no significant effects on motor function by local perinueral injection.3. Local perineural injection of ZD7288had no effects on heart rate, but intraperitonealinjection of ZD7288significantly decreased the heart rate.Main conclusion:1. The degree of conduction failure in polymodal C fibers from CFA rats wassignificantly decreased. 2. We firstly observed that ZD7288enhanced the degree of coduction failure in a doseand activity-dependent manner, but has no significant effects on the conduction of A fibers.These results indicated that ZD7288selectively inhibited the input of nociceptiveinformation.3. ZD7288inhibited mechanical allodynia and thermal hyperalgesia by perineuralinjection but had no effects on motor function and heart rate, which provide experimentalbasis for a new peripheral analgesia methods.4. Ih current in small-sized DRG neurons from CFA rats was significantly increased,and the amplitude and duration of AHP in CFA DRG neurons was remarkedly decreased.The expression of HCN2in small-sized DRG neurons and C fibers was significantlyincreased. These results may contribute to the reduction of conduction failure and CVs.