Nociceptive Innervation and Receptors in the Bladder
Bladder pain syndromes are common and especially prevalent in women. One of the problems in treating bladder pain is that our fundamental knowledge of nociceptive signaling in the bladder is limited. For example, nociceptive innervation in the bladder and the precise localization of key nociceptive receptors are not well defined. There is considerable controversy as to whether TRPV1, a major “pain” receptor, is expressed in non-neuronal tissues such as the bladder urothelium. Non-specific antibody labeling, and the preferential use of male rodents are major limitations of previous studies. The goal of this project is to exploit genetic models to precisely map the TRPV1-lineage and TRPV1 expression in male and female mouse bladders at different developmental stages. Further, we will confirm bona fide function of these channels in identified cells/tissue layers. At the conclusion we will be able to submit to GUDMAP the expression of the TRPV1 gene lineage and TRPV1 expression in bladder relative to specific tissue anchor genes.
Specific Aim 1: To map arteriolar smooth muscle expression of TRPV1-lieage in post-natal bladder.
We will exploit a TRPV1-Cre reporter mouse line that selectively labels (with fluorescent reporter tdTomato) the TRPV1 lineage that includes ~90% of nociceptive nerves, as well as a subset of arteriolar smooth muscle cells. We will quantify the density of TRPV1-lineage expression in ASM in whole bladder mounts, from P2, P7, P14, P28, P90, P200 and P260 day-old male and female mice. This will allow us to obtain a low-resolution map of regional (bladder dome vs. bladder base) distribution of nociceptive innervation at distinct postnatal stages in both sexes. We will perform double labeI (tdTomato-IHC) to co-localize TRPV1-lineage to specific tissue markers (uroplakin 3a, smooth muscle myosin heavy chain, SM22α)
Specific Aim 2: To map TRPV1 expression in neonatal, prepubertal and postpubertal bladder.
To identify real-time expression of TRPV1 we will utilize TRPV1PLAP-nlacZ mice, in which placental alkaline phosphatase (PLAP) and nlacZ are expressed under the control of the TRPV1 promoter. PLAP histochemistry can be used to label cell body and axonal processes of TRPV1-expressing cells. LacZ staining will selectively label non-neuronal TRPV1. We will validate reporters genes using ISH to TRPV1. Analysis of these reporters in thin tissue sections of bladder will reveal spatial-temporal distribution of TRPV1. To precisely map non-neuronal TRP expression we will co-localize expression of the TRP-reporter with gene anchors (uroplakin 3a, smooth muscle myosin heavy chain, SM22α) using IHC.
Specific Aim 3: To identify TRPV1 expression by function in isolated bladder cells.
An important validation of reporter gene expression is to confirm functionality of TRPV1 channels in identified bladder cells. We will isolate and prepare primary cell cultures of 3rd order bladder arterioles. We will assess TRPV1 channel function using Ca2+ imaging, electrophysiology and by monitoring contractile responses. Expression of TRPV1 will be verified using well-defined agonists/antagonists and by the distinct electrophysiological signature of these channels.
Grant number: 1U01DK101040