| Pain is not only a symptom, but also a kind of disease. The incidence of chronic pain is increasing year by year. More and more people suffer from chronic pain, which severely threatens patients’ life quality and physical and mental health. These patients are overwhelmed with chronic pain so that they hardly wish to live. Chronic pain is recurrent and very difficult to treat. In addition to the burden on the individual, chronic pain also exacts a high cost from society[1]. Chronic pain is often accompanied by aversive emotion, such as anxiety, depression, fear and even vicious mood. In recent years, with the intensive research in the cellular and molecular level, the mechanism research of chronic pain acquires the great progress. However, the treatment of pain is not optimistic, currently the analgesic used in clinical treatment is limited to two kinds which is opioid and no steroidal anti-inflammatory drugs, because the former can lead to mental addiction, tolerance and other serious side effects, the use is limited. The latter is not broad-spectrum effective analgesic because the effect is not very good. Therefore, to explore the underlying mechanisms of chronic pain and to develop new analgesic therapy is urgently needed and is the basic responsibilities and goals of each pain researcher and clinician.Transient receptor potential(TRP) channel is the nonselective cation channels super family of calcium transparent which is located on the cell membrane, TRP is divided into six subfamily, these are TRPC, TRPV, TRPM, TRPA, TRPP and TRPML[2]. Accumulating evidence has shown that transient receptor potential(TRP) channels play a crucial role in sensing a variety of external noxious stimuli, such as cold, heat, mechanical and chemical stimuli. These noxious stimuli are transformed into action potentials and transmitted to the spinal cord and then further to the brain which eventually evoke pain[3, 4]. It is well known that TRPV1 is mainly responsible for receive heat and acid stimulation of both inside and outside environment[5].TRPM8 encoding the cell’s perceptions of cold[6]. TRPA1 can be activated by chemical irritants in the cell environment [7]. In pathological conditions, such as injury or inflammation, the up-regulated expression of above channel subtypes and enhancement of plasticity changes will play an important role in the development of chronic pain[3, 4].TRPC channel is the subfamily which is cloned in TRP channel super familyin the first time. However, due to the lack of specific tools, the explorations of function of TRPC channels are far behind other TRP channels. TRPC channels including seven subtypes, according to its similarity and proximity of structure and function which is divided into three subsets: respectively are TRPC1/4/5、TRPC2、TRPC3/6/7[2]. Because channel proteins of the subgroup in structure and function have a great deal of redundancy, we choose TRPC1/4/5 knockout mice to research.Recently, researching a new generation of analgesics which is direct at TRP channels of the source of the development of pain information has strided the pace of history, this kind of analgesics are hoped to eliminate the production and transmission of chronic pain signals, and can prevent the further formation of the central sensitization, achieving the target that the analgesic effect of low toxicity and efficient[3, 8]. Therefore, to explore the underlying mechanisms of chronic pain and to develop new analgesic therapy is urgently needed and is the basic responsibilities and goals of each pain researcher and clinician.Part 1:To investigate the function of TRPC1/4/5 channels in sensing external stimuli under physiological and pathological statesObjective:To observe the function of TRPC1/4/5 channels in sensing external stimuli under physiological and pathological statesMethods : In the normal physiological condition, we detect the difference of reactivity to mechanical and thermal stimulation between knockout mice and wild-type mice. Subcutaneous inject Complete Freund’s adjuvant(CFA) into the unilateral planta pedis of adult mice hind paw to establish the pain model. Then detect the difference of reactivity to mechanical and thermal stimulation between knockout mice and wild type mice in multiple time points. After injecting bradykinin into one side of the hind paw, we detect the difference of spontaneous pain reaction time. To verify whether knockout the TRPC1 /4/5 genes can affect the mice’s motor coordination ability by rotate rod.Results : In physiological state, the reactivity of TRPC1/4/5 knockout mice significantly declined in the response to mechanical stimulation and thermal stimulation compared to the wild type mice. In the condition of chronic inflammatory pain, the reactivity of TRPC1/4/5 knockout mice also significantly declined in the response to mechanical stimulation and thermal stimulation compared to the wild type mice. The response time of spontaneous pain caused by bradykinin obviously decreased compared with wild type mice. Through the rotate rod experiment confirmed that knockout TRPC1/4/5 channel will not affect the mice’s motor coordination ability.Part 2: The mechanism of TRPC1/4/5 channels involved in mediating peripheral sensitizationObjective: To assess the effect of TRPC1/4/5 channels on the excitability of nociceptive DRG neuronsMethods: By using whole-cell patch-clamp recordings in intact DRG preparation,passive and active membrane properties was assessed in small nociceptive DRG neurons derived from wildtype and TRPC1/4/5 TKO mice under basal states and 24 hours after CFA injection.Results:Passive membrane properties including resting membrane potential(RMP) and membrane resistance(Rm) as well as membrane capacitance(Cm) of small DRG neurons tested were not significantly different between wildtype and TRPC1/4/5 TKO mice. However, the active membrane properties of small DRG neurons from TRPC1/4/5 TKO mice represented significant differences compared with wildtype controls. Small DRG neurons from TRPC1/4/5 TKO displayed reduced excitability, manifesting as increased rheobase to evoke actional potential(AP) and reduced AP frequency induced by a depolarizing step current as compared to that from wildtype mice. This result suggests that TRPC1/4/5 channels are involved in determining the excitability of DRG neurons.Passive and active membrane properties were assessed in small nociceptive DRG neurons derived from wildtype and TRPC1/4/5 TKO mice under inflammatory pathological states. Following CFA inflammation, small DRG neurons from wildtype mice become hyperexcitable, manifesting as decreased rheobase to evoke AP and enhanced AP frequency in response to depolarizing current injection. In contrast, TRPC1/4/5TKO mice demonstrated a significantly lower excitability than wildtype mice after CFA inflammation. It can be inferred from the above that TRPC1/4/5 channels are key determinants in the hyperexcitability of nociceptive DRG neurons under pathological states.Part 4: To evaluate the influence of TRPC1/4/5 channel on the expression of c-Fos and p-ERKin the spinal cord induced by irritantsObjective:In order to get further evidence of TRPC1/4/5 channel restrain the pain reaction.Methods : The formalin-induced inflammatory pain models were set up with the above methods. Two hours later, the lumbar enlargement was removed after paraformaldehyde perfusion and fixation, then post-fixed, dehydrated, frozen sectioned and dyed by immunohistochemical ABC method.Result:In the process of formalin induced pathological pain, the expression of c-Fos and p-ERK of L4- L5 segmental spinal cord dorsal horn shallow neurons of the knockout mice obviously down regulation compared with wild type mice.Part 3: The central sensitization mechanism of TRP1/4/5 channel induced pain sensitivityObjective:Further confirmed the cellular and molecular mechanism of TRP1/4/5 channel induced pain sensitivityMethods:Adopt the mice after born 15-21 days, under the normal state and after chronic inflammatory pain models induced by the 24 hours after CFA injection, producing spinal cord slice sample, transverse spinal slices were obtained and whole cell patch clamp recordings of spinal dorsal horn lamina I neurons were performed. Observe the difference of excitability of spinal dorsal horn I layer neurons between knockout mice and wild-type mice. Observe the influence of TRPC1/4/5 channel to the primary afferent synaptic transmission efficiency and the change of plasticity. Record the passivity and active membrane properties of noxious DRG neurons and spinal cord dorsal horn I layer neurons, and record the evoked excitatory postsynaptic currents(e EPSCs) of spinal cord dorsal horn I layer neurons induced by stimulate the noxious primary afferent fibers. In the condition of chronic inflammatory pain, record the spontaneous excitatory postsynaptic currents(s EPSCs) of spinal cord dorsal horn I layer neurons.Results:Under basal states, excitatory synaptic currents(e EPSCs) in spinal lamina I neurons were evoked by electrical stimulation of dorsal root entry zone. Input-output curve(I-O curve) demonstrated that the amplitude of e EPSC increases with the increase of stimulation intensity. As compared to wildtype mice, TRPC1/4/5 TKO mice showed the characteristic right-ward and downward shift in the I-O curve of e EPSCs, reflecting the reduced basal synaptic transmission. Following CFA inflammation, I-O curve of e EPSCs from wildtype mice demonstrated a dramatic left-ward and upward shift over basal curves, reflecting synaptic potentiation. In striking contrast, TRPC1/4/5 TKO mice exhibited a significantly less potentiation of synaptic transmission than wildtype mice after CFA inflammation. We can infer from the above that TRPC1/4/5 channels are involved in the synaptic potentation under inflammatory pathological states.To further elucidate whether the effect of TRPC1/4/5 channels on synaptic potentiation is presynaptic or postsynaptic in origin, we performed two analysis, s EPSCs and PPR analysis. Analysis of s EPSCs revealed that deletion of TRPC1/4/5 channels was able to reduce the frequency and amplitude of s EPSCs in CFA-inflamed mice. This result suggests that TRPC1/4/5 channels may facilitate spinal synaptic transmission and plasticity by presynaptic and postsynaptic mechanisms.In the condition of physiological status and chronic inflammatory pain,Observed the excitatory of DRG neurons and spinal cord dorsal horn neurons of TRPC1/4/5 knockout mice significantly decreased compared with wild type mice, which manifested as decreased action potential(AP) frequency and amplitude, increased AP half-width, threshold and rheobase.Conclusions:1. By utilizing TRPC1/4/5 TKO mouse model, we demonstrated for the first time that TRPC1/4/5 channels play an important role in the basal mechanical and thermal nociception. Furthermore, TRPC1/4/5 channels exert pronociceptive effect under inflammatory pathological states.2. We demonstrated for the first time that TRPC1/4/5 channels can modulate the excitability of nociceptive DRG neurons under basal states. Upon injury or inflammation, TRPC1/4/5 channels mediate peripheral sensitization of nociceptive DRG neurons, which might underlie their pronociceptive effects under patholgocial states.3. TRPC1/4/5 channels play an important role on enhanced activity of superficial dorsal horn of the spinal cord neuron, and the expression of algogenic substance caused by pain and nociceptive stimulation.4. We demonstrated for the first time that TRPC1/4/5 channels make a great contribution in spinal synaptic transmission and synaptic plasticity in spinal dorsal horn. Upon injury or inflammation, peripheral sensitization and spinal synaptic plasticity has been assumed as the cellular basis of chronic pain. TRPC1/4/5 channels exert a marked facilitating effect on spinal synaptic transmission and plasticity. s EPSCs analysis strongly support the inference that the potentiation of synaptic transmission by TRPC1/4/5 channels under inflammatory states comes about via presynaptic mechanisms involving a decrease in release probability and postsynaptic mechanism involving a increase in relativity of receptor. |
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