The Role Of Piezo2 Channels In Peripheral Nerve Sensitization Of Premature Ejaculation

BackgroundSince Schapiro et al.first proposed the concept of premature ejaculation in 1943,the specific definition of premature ejaculation has been controversial.Before,the definition of premature ejaculation was usually organized by various professional organizations or individuals,but these definitions were flawed,non-evidence-based,lack of professional operating standards,excessive ambiguity,and excessive dependence on the subjective judgment of the diagnose.Until 2013,the International Society of Sexual Medicine(ISSM)proposed a multivariate evidence-based definition of lifelong premature ejaculation and acquired PE.The etiology of premature ejaculation is complicated.It was considered that penile hypersensitivity may be one of the main pathologic mechanisms of PE.A case-control study conducted by Xin suggested that PE patients had hypersensitivity in glans penis and penile shaft.Interestingly,with quantitative sensory testing analysis,Salonia et al.found that the PE patients had penile hyposensitivity,instead of penile hypersensitivity.One potential reason for the inconsistency is that most of the studies predated the new ISSM definition of PE and,as such,the definitions of "normal and"PE" are somewhat arbitrary and variable across studies.The relative small sample size also contributed to the conflicts among studies.Therefore,in the present study,we used penile biothesiometer to reassess the correlation between penile hypersensitivity and lifelong PE in a relatively large cohort,as defined by the criteria identified by the ISSM definition of PE.In order to investigate the pathogenesis and pathological process of primary premature ejaculation,we established a rat model of premature ejaculation based on the ejaculation distribution theory in preparation for further study for the mechanisms of peripheral nerve sensitization in premature ejaculation.The Piezo channel is a new class of mechanically sensitive ion channel first discovered by Coste et al.in 2010.They found that the Piezo protein has completely independent mechanical sensitivitye,which was considered as a milestone in the field of neurobiology.Although the role of the Piezo2 channel in normal tactile sensation has been affirmed,and the role of the Piezo2 channel in various mechanical susceptibility was also confirmed.However,the physiological role,especially the peripheral sensory nerve sensitization of premature ejaculation,has not been elucidated.In present study,we established a rat model of premature ejaculation by natural screening,and confirmed the presence of Piezo2 channel on the nerve endings of the penis and the DRG of the penis.Then further explored the possible neural mechanisms of Piezo2 channel expression and functional changes in DRG by means of molecular biology and electrophysiology as well as the mechanisms of penile sensitivity to mechanical stimulation(peripheral sensitization).Part I Significance of penile hypersensitivity in premature ejaculation Purpose:To assess the correlation between penile hypersensitivity and premature ejaculation.Methods:1.A consecutive 420 subjects attending our andrologic clinic were evaluated for suspected PE.After the initial sexological assessment,213 participants of primary PE were included.2.Pre-assessment of intravaginal ejaculatory latency time(IELT)by stopwatch was measured for the 4-week baseline period during which all participants were asked to have sexual intercourse at least four times.According to the IELT,the Lifelong PE participants were divided into two groups:mild PE group included 152 participants with IELT?30s and?60s and severe PE group included the other 61 participants with IELT<30s.The remaining 124 men did not fulfill the criteria for a diagnosis of PE were considered as controls.3.Penile skin vibratory thresholds were comprehensively assessed and recorded by means of biothesiometer.Results:1.The characteristics of the participants:Patients and controls were comparable with regard to age,BMI,tobacco smoking,alcohol abuse and IIEF-5 scores.When comparing the vibratory thresholds between the groups,all changes in glans penis and penile shaft were statistically significant.2.The correlation between vibratory thresholds and IELT:(1)In the control group,there was no statistically significant correlation between penile vibratory threshold and IELT in glans penis or penile shaft(glans penis:rs=0.127,P=0.161;penile shaft:rs=0.156,P=0.084).In the PE group,there was a significant positive correlation between penile vibratory threshold and IELT in glans penis or penile shaft(glans penis:rs=0.349,P=0.0001;penile shaft:rs=0.341,P=0.0001).(2)In the mild PE group,there was a significant positive correlation between penile vibratory threshold and IELT in glans penis or penile shaft(glans penis:rs=0.315,P=0.0001;penile shaft:rs=0.27,P=0.001).(3)In the severe PE group,there was a significant positive correlation between the penile vibratory threshold and IELT in glans penis or penile shaft(glans penis:rs=0.389,P=0.002;penile shaft:rs=0.354,P=0.005).3.The ROC curve of penile sensory threshold as sensitivity and specificity for predicting the severity of premature ejaculation:(1)The cut-off of glans thresholds value for PE was 4.25 V(AUC:0.852,95%CI:0.813-0.892).This value had a sensitivity of 73.4%and a specificity of 83.6%for differentiating the status of PE.(2)The shafts thresholds value<4.15 V was a potential cut-off level for the prediction of the severity.The value had an AUC of 0.893(95%CI:0.895-0.927),with a sensitivity of 77.4%,and a specificity of 86.9%.Conclusion:Penile peripheral nerve in patients with lifelong PE is sensitized,Penile hypersensitivity appears to be a major factor leading to shorter IELT in patients.In addition,penile vibratory threshold is a valid measurement tool to predict the severity of PE.Part ? Establishment of a rat model of primary premature ejaculationPurpose:To investigate the feasibility of establishing a rat model of primary premature ejaculation using the ejaculation distribution theory by natural screening.Methods:1.85 male Wistar rats and 52 female Wistar rats were selected.The females were first surgically castrated.Then,estrus were induced by subcutaneous injection of 20 ?g estradiol benzoate at 48 h and 500 ?g oprogesterone and 4 h(all dissolved in 0.1 ml of sesame oil)before the mating experiment.2.The screening experiment time was 19:00-21:00.Male rats and hormone-induced female rats were caged 1:1.The observation experiment was carried out 6 times,once a week,and the last 3 times mating process were recorded.3.We observed the male animals for mounting latency(ML),intromission latency(IL),ejaculation latency(EL),mounting frequency(MF),and ejaculation frequency(EF).4.Male rats with mean EF<1 were classified as "sluggish ejaculation",male rats with 1<mean EF ? 2.5 were classified as "normal ejaculation",and male rats with EF mean>2.5 were classified as "rapid ejaculation”.Results:Finally,the last three mating processes of 134 male rats were successfully recorded.Among them,104 male rats with "normal ejaculation" had an average ejaculation frequency of 2.07±0.74;14 male rats with "sluggish ejaculation" had the average ejaculation frequency of 0.63±0.42;16 male rats with "rapid ejaculation" had the average ejaculation frequency of 4.04±0.56.The EL was significantly shorted and theMF was significantly decreasedin the "rapid ejaculation" group than in the "sluggish and normal ejaculation" groups.No statistically significant differences were observed in the ML among the three groups of rats.Conclusion:Based on the mean ejaculation frequency,the male rats with "rapid ejaculation" were effectively screened,and this animal model may play an important role for further study of primary premature ejaculation.Part ? Piezo2 channel in penile DRG neurons may involve in the pathogenesis of premature ejaculationPurpose:To investigate the expression and function of the Piezo2 channel in the DRG neurons of the penis in PE rats using molecular,electrophysiological and the sexual behavior methods.Methods:1.Labeling and separation of DRG:DRG was retrogradely labeled by Dil firstly.10-14 days later,the DRGs from the L6-S1 were removed under the microscope surgically.2.Immunehistochemistry was used to observe the expression of Piezo2 channel on the nerve endings of the penis and the DRG of the penis.3.The expression of the Piezo2 channel in the DRGs of the two groups of rats was detected by immunohistochemical quantitative analysis and RT-PCR after removing the labeled DRGs from L6-S1.4.Calcium imaging technique:Firstly,The Dil-labeled DRG neurons were identified.Then,the microelectrode was approached to the cell at a 45-degree angle and a 4um displacement was given to the cell,and the intracellular calcium signal of DRG neurons was recorded during stimulation.5.Patch clamp technique:The microelectrode was approached to the cell at a 45-degree angle and a 4um displacement was given to the cell,and the inward current was recorded during the stimulation under MP-285 miniature operating system.6.Inward current induced by mechanical stimulation blocked by FM1-43:after the first mechanical stimulation of the cells,one specific blocker of the piezo2 channel FM1-43(10 uM)was administered for 5 minutes,and then a second mechanical stimulation was given.The inward currents before and after FM1-43 were recorded.7.Piezo2 oligodeoxynucleotide interference:The pre-screened normal rats were divided into control group,missense oligodeoxynucleotide group and antisense oligodeoxynucleotide group,which were labeled with Dil dye,then the intrathecal tube was injected into the space between L6-S1,and the oligodeoxynucleotide was injected with a micro syringe..8.Immunohistochemistry and PCR detection after oligodeoxynucleotide interference:Immunohistochemical quantitative analysis and PCR were detected in oligodeoxynucleotide-interfering rats according to protocol above to confirm the effectiveness of oligodeoxynucleotide interference.9.Patch clamp study after oligodeoxynucleotide interference:At the same time,the patch clamp detection will be performed according to the protocol above,after the oligodeoxynucleotide interference model constructed.10.Behavioral observation of rats after antisense oligodeoxynucleotide interference:An oligodeoxynucleotide interference model was constructed on pre-screened ejaculation rats,and the changes in sexual behavior indicators after antisense oligonucleotide interference were observed.Results:1.Immunohistochemistry study showed the Piezo2 channel was abundantly expressed in rat penile glans nerve endings and DRGs of L6-S1.2.Immunohistochemical quantitative analysis and the mRNA expression of Piezo2 channel in DRG of PE rats was significantly higher than that of normal rats.The difference was statistically significant(P<0.01).3.The intracellular calcium elevation induced by mechanical stimulation under the same intensity in PE rats(2.62±0.61)was greater than that in normal rats(1.84±0.53).The difference was statistically significant(P=0.004).4.Mechanical stimulation induced a rapidly activated and rapidly inactivated inward current that could be blocked by the Piezo2 channel specific blocker FMI-43(n=8,P<0.01).The inward current density induced by mechanical stimulation in PE rats was 15.12±5.03pA/pF(n=11),which was significantly higher than that of normal rats 9.78±3.52A/pF(n=9).The difference was statistically significant(P<0.01).5.The activation time to peak of PE rats(n=12)was 91 5±150ms,the time of decay to 80%peak was 670±200ms.The activation time to peak of normal rats(n=10)was 680±110ms,the time of decay to 80%peak was 5 10±150ms.There was no significant difference in the activation and inactivation characteristics of mechanically induced currents between the two groups.6.Immunohistochemical quantitative analysis and PCR detection showed that the expression of Piezo2 channel in the antisense oligodeoxynucleotide group was significantly lower than that of the missense oligodeoxynucleotide group and the control group(P<0.01).7.Patch clamp showed a mechanical stimulation current density of 3.77±1.40 A/pF in the antisense oligodeoxynucleotide group and 9.81±4.12 pA/pF,9.08±4.94 pA/pF in the control and missense oligodeoxynucleotide groups,respectively.,there were significant differences in the three groups(P<0.01).At the same time,there was no significant difference in the activation and inactivation characteristics of mechanically induced currents in the three groups(P>0.05).8.Compared with the rats in the missense oligodeoxynucleotide group,the ejaculation latency(EL)of the antisense oligodeoxynucleotide group was significantly prolonged,the mount frequency(MF)was significantly increased,the ejaculation frequency was significantly reduced(P<0.01).Conclusion:Piezo2 channel was abundantly expressed in rat penile glans nerve endings and DRGs from L6-S1.The expression of Piezo2 channel was up-regulated in DRGs in PE rats,and the mechanical stimulated current density was significantly enhanced.The currents could be blocked by the Piezo2 channel specific blocker FMI-43,revealing that the Piezo2 channel plays a major role in peripheral nerve sensitization in rat penis.