HCN2 Involved In Peripheral Neuropathic Pain And The Generation Of It’s Conditional Knockout Mice

Neuropathic pain is caused by a lesion or disease of the somatosensory system.The clinical symptoms of neuropathic pain is spontaneous pain,hyperalgesia and allodynia.Many diseases can contribute to neuropathic pain,such as diabetes,herpes zoster,leprosy,HIV infection and radiculopathy.According to epidemiological studies,the prevalence of neuropathic pain in the general population has been estimated to be in the range of 6.9%-10%.In this situation,there could be 97-140 millions people suffering from neuropathic pain.The mechanisms of neuropathic pain is unclear because the factors that cause neuropathic pain are complex.Currently,Studies on the mechanism of neuropathic pain are mainly concentrated in the changes in ion channel,function and expression,changes in second-order nociceptive neuronal function and changes in inhibitory interneuronal function.Hyperpolarization-activated cyclic nucleotide-gated(HCN)cation channels belong to the superfamily of voltage-gated pore loop channels.HCN channels are encoded by HCN1-4 gene family and have four subtypes.These channels are activated upon hyperpolarization of membrane potential and conduct an inward,excitatory current Ih in the nervous system.Ih current involved in a variety of neurological activities,including the control of resting membrane potential,induced nerve oscillation,regulation of neurotransmitter release.Studies have shown that HCN channels are widely distributed in pain conduction pathways and play a vital role in the formation and development of pain.Inflammatory mediator PEG2 can enhance the effect of HCN2 deletion on the action potential of small sensory neurons,and most of these small sensory neurons belong to pain receptors,which suggests that HCN2 plays an important role in inflammatory pain.At the same time,Neuropathic pain is reduced by deletion of adenylate cyclase 5 and also by inhibition of COX2 or by deletion ofm PGES-1,two enzymes that are responsible for the biosynthesis of PGE2.These observations suggest that the proposed mediator may be PGE2.The work reported here suggests that HCN2-selective blockers may have value as analgesics to combat both inflammatory and neuropathic pain.Therefore,this study will use the spinal nerve ligation injury(SNL)model to confirm whether HCN2 is involved in peripheral neuropathic pain,and as far as possible studying its mechanism.Part oneObjective: Using the spinal nerve ligation model of neuralgia to study whether HCN involved in peripheral neuropathic pain.Content: 1.To compare the success rate of spinal nerve ligation model under different surgical methods,and evaluate whether the model meets the requirements.2.The pharmacological method was used to detect whether the non-specific inhibitor ZD7288 of HCN2 had the effect of reversing pain.Thus indirectly demonstrate whether HCN2 is involved in peripheral neuropathic pain.3.q PCR method was used to detect the transcription of HCN2 in DRG of neuralgia model rats.4.Western blot was used to detect the expression of HCN2 protein in DRG of neuralgia model rats.Methods: 1.Using three types of surgical methods to establish neuralgia model:1)Sham group,only exposing the spinal nerve;2)SNL group,with a silk suture,L4 SN without treatment;3)m SNL group,loose ligate L4 SN with a chromic gut suture,tightly ligate the L5 SN;4)SNA group,cut the SN at the distal end of ligtion on the basis of m SNL.The rats were subjected to behavioral experiments a week after surgery.2.The neuralgia rats were randomly divided into three groups(n=6): 1)negative group,0.9% physiological saline(500 μl/kg),intra-plantar injection of left hindpaws;2)positive group,gabapentin(10 mg/kg)diluted in 0.9% physiological saline,intraperitoneal injection;3)experimental group,ZD7288 diluted in 0.9% physiological saline(15μg/kg),intra-plantar injection of left hindpaws.The behavioral experiments were tested before injection and 1h,4h,24 h and 48 h after injection.3.Obtain the dorsal root ganglion of neuralgia rats.Extracting total RNA and c DNA was obtained by reverse transcription.And then using real-time quantitative PCR to detect the changes of HCN2 m RNA.4.Obtain the dorsal root ganglion of neuralgia rats.Using western blot to detect the changes of HCN2 protein.Results: 1.1.The rat model of spinal nerve ligation was successfully established.2.ZD7288 can reverse the pain symptoms of SNL rats as well as Gabpentin.3.The m RNA of HCN2 was significantly increased in the DRG(P<0.01)4.The protein of HCN2 also increased in SNL rats’ DRG(P<0.05)Conclusion: HCN2 is involved in the pathogenesis of neuropathic pain,and its mechanism will provide a theoretical basis for the treatment of neuropathic pain.Part twoObjective: Establishing the knockout mice of HCN2,and using it as a research tool to study the mechanism of HCN2 involved in peripheral neuropathic pain.Content: 1.According to the mechanism of Cre/Lox P recombinase system,two rounds of hybridization of mice with homozygous mice of hcn2 gene and NSE-Cre mice were used to obtain HCN2 knockout mice.2.Using behavioral experiments to carry out basic phenotypic analysis of HCN2 knockout mice.Methods: 1.Firstly,hcn2flox/flox mice was obtained by selfing of hcn2flox/+mice;secondly,hcn2flox/+·cre+ mice was obtained by hybridizing with hcn2flox/flox mice and NSE-Cre mice;thirdly,hcn2 flox /flox· cre+ mice was obtained by hybridizing with hcn2flox/flox mice and hcn2flox/+·cre+ mice.The genotype of mice was identified by PCR method using mice tail DNA.2.Using immunofluorescence method to confirm the effect of HCN2 gene knockout.3.Comparing the appearance and weight of normal mice and HCN2 knockout mice to evaluate it’s capacity of growth and development.4.Using open field experiment,footprint experiment and roller experiment to evaluate the behavioral ability of HCN2 knockout mice.5.Using hot plate experiment to evaluate the heat sensitivity of HCN2 knockout mice.Results: 1.Successfully breed hcn2 flox /flox· cre+ homozygous mice.2.Immunofluorescence results show that HCN2 is partially knocked out in the HCN2 knockout mice.3.There was no significant difference in the growth and development ability between HCN2 knockout mice and normal mice.4.The results of behavioral study showed that there was no significant difference in behavioral ability between HCN2 knockout mice and normal mice.5.The results of hot plate experiment showed that there was no significant difference in heat sensitivity between HCN2 knockout mice and normal mice.Conclusion: We successfully constructed HCN2 conditional knockout mice.HCN2 in the mice has not been completely knocked out.The reason remains to be analyzed.Although the knockout effect did not meet our expectations,the mouse will have a certain value.It still can be used for neuropathic pain research.