We enquired whether the assumed inverse relationship between transepithelial resistance (TEER) and bladder permeability is attested by IVIVC analysis,  which is mandated by regulatory agencies for determining the true permeation rate of drugs. The purported significance of high TEER >3000ohm/cm^2 of excised human urothelium is refuted by the ground truths of  >20% dose absorption of intravesical Oxybutynin by healthy human volunteers having intact urothelium (ref.1) and significant bladder 
absorption of radionuclide during direct radionuclide cystography (DRC) (ref.2).  The discordance between clinical facts and pervasive dogma of "water-tight" bladder implicate an integral role of urothelial perfusion in the systemic uptake of instilled oxybutynin(357.486 Daltons), which we investigated by  instilling paramagnetic dye, Gadobutrol (604.71 Daltons) (ref.3)  as a proxy for permeation of Oxybutynin to detrusor layer of rat bladder with(in-vivo) or without (ex-vivo) intact perfusion.

Under isoflurane anesthesia, 3-4 months old female Sprague-Dawley rat bladder(n=9) were briefly exposed to 0.5mL of protamine sulfate 10mg/mL for 30min prior to 0.3mL aqueous instillation of Gadobutrol 4mM and/or Ferumoxytol 5mM mixture with 24Gauge angiocatheter for intravesical contrast enhanced (ICE)-MRI. Bladder was excised or left intact in animal for T1 weighted Turbo spin echo scans at 7 Tesla scanner. The excised bladder was encased in gelatin gel for ICE-MRI and cryosections were stained for histology.

Rat urothelium (*), injured by protamine is indistinguishable from urine (Fig.1A), urothelium is brightened after urine is replaced by Gadobutrol and Ferumoxytol(731 KDa) mixture in-vivo (Fig.1B) and ex-vivo (right image, Fig.1C). The absence of bright urothelium (*) post- instillation of Ferumoxytol without Gadobutrol (left image, Fig.1C) implies that lumen darkened by luminal retention of larger sized Ferumoxytol casts urothelium (*) brightened by paracellular diffusion of smaller Gadobutrol in sharp relief. An integral role of perfusion in bladder permeability is consistent with bright detrusor (#) only seen ex-vivo (Fig.1C) but not in bladder with intact perfusion, in-vivo (Fig.1B). The absence of breathing and bowel motion ex-vivo (Fig.1C)  increases the pixel resolution 5x of in-vivo bladder (Fig 1B).

The vascular architecture of mucosa showcased in rat bladder wall histology (Fig.1D) has the capacity for mucosal perfusion rate of ~0.6mL/min to dilute the diffused Gadobutrol and restrict its direct diffusion to detrusor, which however occurs in absence of perfusion, ex-vivo. Mucosal perfusion rapidly washes away diffused radionuclides, drugs and dyes from urothelium to sustain a steeper concentration gradient for driving a faster Fickian diffusion (in-vivo) flux than the flux of radionuclides and drugs predicted ex-vivo (ref.2). Therefore, bright urothelium and persistently dark detrusor in vivo (Fig.1B) embodies an exponential concentration gradient of instilled dyes, drugs (Oxybutynin) and radionuclides in DRC from urothelium to detrusor (ref.1-2). Instilled Gadobutrol in ICE-MRI mimics Evans blue dye and Fluorescein in brightly staining the umbrella cell borders (paracellular diffusion) but not the apical surface of umbrella cells and detrusor, in vivo.  As a result, ICE-MRI in vivo is consistent with ten-fold higher systemic uptake of radionuclide and of lidocaine from human bladder than from human vagina, even though TEER of excised urothelium is tenfold higher than TEER of excised vagina, 3000 vs 250ohm/cm^2.

The IVIVC analysis of ICE-MRI is consistent with an integral role of urothelial perfusion in bladder permeability, which cannot be studied on excised bladder sheets with Ussing chamber. Persistently dark detrusor of rat bladder in ICE-MRI in-vivo implies that a 100 fold faster perfusion rate of human bladder mucosa accelerates the paracellular diffusion of drugs, dyes and radionuclides which culminates in >20% intravesical dose absorption of instilled lidocaine and oxybutynin. The IVIVC paradigm can help resolve bladder permeability controversy by authenticating the conclusions drawn from ex-vivo setup with in-vivo ground truths .

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