| Background and ObjectivePain is an unpleasant feeling and emotional experience.Due to the different locations and causes,pain can be divided into different types such as cutaneous pain,visceral pain,muscular pain and neuropathic pain,etc.These stubborn pains affect the health of more than one-sixth of the world's population and cause a great burden to society.Neuropathic pain and visceral pain are common types of pain in people's daily life.However,due to the complexity of pain mechanisms which including both peripheral and central mechanisms,the treatment of pain,especially chronic pain is a clinically difficult problem.At present,we know that the input of pain signals is regulated at multiple levels.At the spinal cord level,there is a presynaptic regulation between the different peripheral afferent signals(such as cutameous A and C fiber afferent,muscular A fiber afferent)and the nociceptive pain signal.For example,as early as 1965,Melzack and Wall's pain gate theory suggested that there is a gate loop between peripheral A fibers and inhibitory SG cells,projection neuron T cells,and C fibers,which regulates the afferent of pain signals.Our previous studies also proved that the lack of afferent peripheral A fiber is a major cause of neuropathic pain.Accordingly,one of the purposes of our study is to clarify the exact regulatory role of cutaneous A fiber afferents and muscular A fiber afferents in neuropathic pain.In addition,our previous study also showed that,cutaneous C fiber afferent has a certain inhibitory effect on muscular C fiber afferent in the condition of inflammatory muscle pain.This inhibitory effect is beneficial for relieving inflammatory muscle pain.This is the regulation of nociceptive stimulation of the body surface on deep muscle pain.So is there such a regulated relationship between cutaneous afferent and visceral pain?Can cutaneous afferent regulate visceral pain?What are the anatomical and physiological mechanisms leading to this phenomenon?The other purpose of our research is to explore the influence of cutaneous afferents on visceral pain and the anatomical basis and physiological mechanism.Materials and MethodsThis study used adult Sprague-Dawley(SD)female rats weighing between 160-220 g and pirt-Gcamp3 mice.The acute injured nerve model(acutely transect the branches of the sciatic nerve:tibial(both muscular and cutaneous),gastrocnemius-soleus(muscular only),sural nerve(cutaneous only))and the tibial nerve venom injection model was used.Immediately after injury,Von Frey hair was used to measure the hind paw threshold,and we observed the behavioral changes of spontaneous pain.Intravenous injection of Evan's blue dye solution was used to observe the exudation of the receptive field on the skin of the hind paw and we also monitored the skin temperature changes of the rats before and after the injury.We recorded the mechanical pain threshold and spontaneous discharge of nociceptive neurons in L4 and L5 dorsal root ganglion(DRG)before and after injury.We also used GCaMP3 mice to observe the changes of calcium activity in DRG neurons before and after nerve injuries.Secondly,we conducted in vivo electrophysiological recordings of L6 and S1 DRGs in a rat colitis model,and observed nociceptive neurons that respond to peripheral cutaneous stimulation and colon expansion.And we also recorded the spontaneous discharges of C nociceptive neurons that respond to different cutaneous stimulation.We next used three chemically induced visceral inflammatory pain models(colitis,cystitis,and gastritis),respectively injected the fluorescent dyes DiI and CTB488 to observe whether there are nociceptive nerves that innervate both the internal organs and the cutaneous receptive field.The Evans blue extravasation also observed in these three models.Results1.Acute transection of the gastrocnemius-soleus and tibial nerve or the injection of venom into the tibial nerve induced tactile pain and spontaneous pain in the adjacent area of the hind paw,but acute transection of the sural nerve or injection of snake venom did not induce pain related behavior.2.Injury to muscular nerve but not cutaneous nerve,caused the decline of mechanical threshold and the rise of spontaneous discharges of nociceptive neurons.(1)After acute transection of gastrocnemius-soleus nerve and tibial nerve or tibial nerve injection with snake venom,the mechanical threshold of nociceptive neurons in L4 or L5 DRGs decreased,and the spontaneous discharge occurred.(2)After the gastrocnemius-soleus and tibial nerve were transected,the fluorescence intensity of neurons in L4 or L5 DRGs of Gcamp mice was significantly increased,which was significantly different from that of sural nerve transection.3.After the tibial nerve was injured,the skin temperature of the lateral and middle receptive fields of the rat's hind paw increased significantly,while the other two nerve branches were injured without significant changes.4.After the tibial nerve was injected with snake venom,the skin temperature of the rat's hind paw increased significantly,while the snake venom injected with the sural nerve did not change significantly.5.After the acute injury of gastrocnemius-soleus and tibial nerve or tibial nerve injection of snake venom,Evans blue extravasation appeared in the receptive field of the hind paw in the rat.6.The effect of different cutaneous afferent stimuli on the spontaneous discharge of DRG neurons in colitis model rats(1)Cotton sweeping on the cutaneous receptive field area can immediately reduce spontaneous discharge,while sweeping in the non-receptive field has no effect.(2)Clamping stimulation on the cutaneous receptive field area can immediately increase the frequency of spontaneous discharge,and sustain at least 10 minutes after stimulation;clamping stimulation on the cutaneous non-receptive field can,to some extent,inhibit spontaneous discharge.(3)Acupuncture stimulation in the cutaneous receptive field can immediately increase the frequency of spontaneous discharge,and sustain at least 10 minutes after stimulation;acupuncture stimulation on the non-receptive field has no effect on spontaneous discharge.(4)Applying capsaicin to the cutaneous receptive field can slowly increase the frequency of spontaneous discharge,and can maintain for at least 10 minutes;applying capsaicin to the non-receptive field has no effect on the spontaneous discharge.7.There are neurons in DRGs that simultaneously innervate both visceral organs(colon,bladder,stomach)and cutaneous skin.(1)The neurons that simultaneously innervate the colon and cutaneous skin are mainly concentrated in L6,S1 and S2 DRGs,and there are no more than 6 dual labeled neurons in each DRG.(2)The neurons that simultaneously innervate the bladder and body cutaneous skin are mainly concentrated in L6,S1 and S2 DRGs,and there are no more than 10 dual labeled neurons in each DRG.(3)The neurons that simultaneously innervate the stomach and cutaneous skin are mainly concentrated in the T7-T11 DRG,and there are no more than 3 dual labeled neurons in each DRG.8.The mechanical threshold of abdominal skin in colitis model rats is declined,and the response to colorectal distension is enhanced.9.Evan's blue extravasation appears in the corresponding cutaneous area of the rat models of colitis,cystitis and gastritis.Conclusion1.Muscular A fiber afferent plays an important role in the regulation of pain signals,and the dorsal root reflex is involved in this process.The lack of muscular A fiber afferent causes changes in calcium of DRG neurons,triggering the spontaneous activity of neurons and mechanical sensitization,and neuropathic inflammation observed in the receptive field.But the lack of cutaneous A fiber afferent does not cause any of the above changes.2.It was found and confirmed that a small number of nociceptive DRG neurons have axon bifurcation in rats,indicating that peripheral neurons can receive pain signals from the skin and internal organs at the same time,and the afferent signals of visceral pain can retrogradely egress to the peripheral skin receptive field,causing inflammatory exudation.It indicates that the pain in a specific cutaneous area may be used as a "window" for the observation of internal organs.3.Applying various noxious or non-noxious stimuli to the cutaneous receptive field and its surroundings may have varying degrees of regulation on visceral inflammation pain... |
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