Upregulation Of Piezo2 Channel In Primary Sensory Neurons By NGF Mediates Mechanical Sensitive Pain In IC/BPS Model Rats

BackgroundInterstitial cystitis/Bladder pain syndrome(IC/BPS)is a chronic pelvic pain syndrome related to the bladder,usually accompanied by urgency or frequency of urination.IC/BPS associated pain is mainly induced by bladder filling and is alleviated after urination,so the pain exhibits obvious mechanical sensitivity.Since bladder filling in normal people does not cause pain,thus IC/BPS associated pain should belong to mechanical allodynia.However,the underlying mechanisms for this mechanosensitive pain is unclear yet.It has been shown that peripheral sensory sensitization is an important pathological mechanism of IC/BPS pain.The upregulation of the expression and function of sensory ion channels such as TRPV1,TRPA1 and TRPV4 in bladder sensory neurons are involved in the peripheral sensitization,and finally leading to IC/BPS associated pain.Peripheral sensitization is also an important pathophysiological mechanism for urgency or frequency(i.e.overactive bladder,OAB).Piezo2 is a mechanical sensing ion channel discovered by Coste and Patapoutian et al in 2010.Because of this finding,Patapoutian won the 2021 Nobel Prize in Medical Physiology.A recent study by Patapoutian et al.demonstrates the important role of Piezo2 in bladder filling sensation.Piezo2 is mainly expressed in the DRG neurons innervating the bladder as well as umbrella cells of the bladder mucosa.Piezo2 Knockout mice showed longer voiding intervals and impaired bladder compliance.But the role of Piezo2 in pathological conditions such as bladder pain and OAB are not clear yet.Recent studies have shown that Piezo2 channels are also expressed in the nociceptors in addition to the non-nociceptive sensory neurons,and is involved in nociceptive mechanical sensation and mechanical allodynia under pathological conditions including inflammation and neuropathic pain.However,no study reported whether Piezo2 contributes to IC/BPS associated mechanically-sensitive pain.Increased in nerve growth factor(NGF)in the bladder is recognized as a key mechanism for IC/BPS associated pain.Recent studies on inflammatory skin pain and bone pain indicate that Piezo2 channel is another downstream target of NGF.Piezo2 channel knockdown attenuated the role of NGF in peripheral sensitization.However,whether the role NGF in peripheral sensitization is associated with up-regulation of Piezo2 in IC/BPS conditions has not been reported.ObjectiveThe main purpose of our study is to examine:(1)the role of Piezo2 channel in IC/BPS associated pain;(2)the role of Piezo2 channel in IC/BPS associated OAB;and(3)the role of NGF in piezo 2 regulation and the underlying cellular pathways.Methods1.Intraperitoneal injection of CYP in rats to establish the IC/BPS model;2.Knocking down Piezo2 mRNA expression in sensory afferents by intrathecal injection of its Antisense oligonucleotides(Antisense).Rats were divided into four groups:control,CYP,CYP+Antisense and CYP+Mismatch;3.Immunofluorescence combined with DiI retrograde labeling to examine the expression of piezo 2 on the DRG neurons innervating the bladder.4.Bladder whole-mount immunofluorescence to examine the piezo 2 expression in sensory nerve endings in bladder mucosa.5.Fluorescence in situ hybridization(FISH)for mRNA was used to quantitatively compare the piezo 2 mRNA expression level in DRG neurons from four group of rats.6.Western Blot was used to quantitatively compare the piezo 2 protein expression level in L6-S1 DRG neurons from four group of rats.7.Ca2+ imaging technique was used to compare the piezo 2 functional expression level in L6-S1 DRG neurons from control and CYP rats.8.Mechanical pain threshold was measured with Von-Frey filaments at lower abdominal region to observe the difference in pain behaviors in four group of rats.9.Bladder pressure-volume measurement(CMG)and urine spot tests were used to observe the difference in voiding behavior in four group of rats.10.Immunofluorescence method was used to observe co-expression of Piezo2 with TrkA,the high affinity NGF receptor,in DRG neurons innervating the bladder.11.Calcium imaging was used to observe the effect of NGF on mechanical stimulation evoked Ca2+increase in bladder DRG neurons.The potential role of PKC,PI3K and ERK1/2 pathways were tested with their specific blockers.Results1.Compared with control rats,CYP rats manifested mechanical hyperalgesia which was presented as increased pain responses in lower abdomen to Von Frey stimulation.CMG recording showed that CYP rats had shortened voiding interval and reduced pressure threshold of the voiding reflex.The urine spot test showed that CYP rats had increased voiding frequency and decreased voided volume,suggesting the presence of OAB.The CYP bladder mucosa showed obvious congestion and edema.HE staining showed mucosa ulcer and edema.All these findings indicate that the IC/BPS model has been successfully established.2.Piezo2 channel expression was found in the cell bodies of the sensory neurons innervating bladder as well as on the bladder nerve endings including TRPV1,CGRP and IB4-positive sensory nerves.3.FISH experiments showed:compared with control rats,rats from CYP and CYP+Mismatch groups had an increased Piezo2 mRNA expression in bladder DRG neurons,while Piezo2 mRNA level was reduced in rats from CYP+Antisense group.4.WB experiments showed:compared with control,rats from CYP and CYP+Mismatch groups had an increased Piezo2 protein expression in L6-S1 DRGs,while Piezo2 protein level was reduced in rats from CYP+Antisense group.5.Ca2+imaging showed:mechanical stimulation evoked increase in intracellular Ca2+ in DRG neurons,which was inhibited with GsMTx4(unspecific Piezo2 antagonist)as well as by knocking down Piezo2 expression suggesting activation of Piezo2 mediated Ca2+ increase.Compared with control,bladder DRG neurons in CYP rats exhibited increased intracellular Ca2+ increase evoked by mechanical stimulation,while it is decreased in CYP+Antisense rats.6.Pain behavior measurements showed:compared with control,rats from CYP or CYP+Mismatch group manifested enhanced responses in low abdomen to Von Frey filament stimulation indicating mechanical hyperalgesia,while the hyperalgesia was reduced in CYP+Antisense rats.7.CYP recording and urine spots test showed:compared with control rats,rats from CYP or CYP+Mismatch group had a shorter voiding interval,increased voiding frequency and reduced pressure threshold for voiding suggesting overactive bladder.while all the presentations were decreased in CYP+Antisense rats.8.Piezo2 channels are co-expressed with the TrkA receptors,the high affinity receptor of NGF,in bladder DRG neurons.Incubation of DRG neurons with NGF significantly increased mechanical stimulation evoked intracellular Ca2+ increase.The blocker of ERK(U0126)pathway significantly reduced NGF induced enhancement in mechanical stimulation evoked Ca2+ increase.ConclusionThe upregulation of Piezo2 channel in bladder sensory neurons is involved in mechanical sensitive pain and overactive bladder in IC/BPS model rats.Increase of NGF in IC/BPS bladder may be the critical factor for Piezo2 up-regulation.Blocking or downregulate Piezo2 channels in sensory neurons may be a new strategy for the relief of mechanical sensitive pain or overactive bladder in patients with IC/BPS.