Atsushi Sengiku

Intravital imaging of mouse urothelium reveals activation of extracellular signal‐regulated kinase by stretch‐induced intravesical release of ATP

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal‐regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two‐photon excitation microscope and a vacuum‐stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch‐induced ERK activation was reproduced in an hTERT‐immortalized human urothelial cell line (TRT‐HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch‐induced ERK activation in TRT‐HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP‐induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor‐expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks.

Feedback on baseline use of staging images is important to improve image overuse with newly diagnosed prostate cancer patients.

BACKGROUND The objective of this study was to evaluate baseline use and positive rates of staging images (bone scan, CT) in newly diagnosed patients with prostate cancer (PCa) and to improve staging image overuse. MATERIALS AND METHODS This retrospective study covered a consecutive series of patients with PCa who underwent stage imaging at our institution between 2006 and 2011. Various clinical and pathological variables (age, PSA, biopsy Gleason score, clinical T stage, positive biopsy core rate) were evaluated by multivariate logistic regression analysis for their ability to predict a positive staging image. All patients were stratified according to the NCCN risk stratification and positive rates were compared in each risk group. RESULTS 410 patients (100%) underwent a bone scan and 315 patients (76.8%) underwent a CT scan. Some 51 patients (12.4%) had a positive bone scan, clinical T3 and T4 being significant independent predictors. Positive bone scan rates for low-, intermediate-, high-, and very high-risk groups were 0%, 0%, 8.25%, and 56.6%. Some 59 (18.7%) patients had a positive CT scan, with elevated PSA and clinical T3, T4 as significant independent predictors. Low-, intermediate-, high- and very high-risk group rates were 0%, 0%, 13.8% and 80.0%. CONCLUSIONS The incidences of positive staging image in low- and intermediate- risk group were reasonably low. Following feedback on these results, staging in low- and intermediate- risk groups could be omitted.

Augmented Reality Navigation System for Robot-Assisted Laparoscopic Partial Nephrectomy

We have developed a surgical navigation system for robot-assisted laparoscopic partial nephrectomy (RALPN). In this system, a three-dimensional computer graphics (3DCG) model generated from each patient’s computed tomography image is overlaid on the endoscopic image, and we control it manually to navigate the vascular structure and tumor location in real time. The position and orientation of the 3DCG model is calculated from the optical flow of the endoscopic camera images, which enables the model to move semi-automatically. We conducted 20 navigations for RALPN from April 2014 to December 2016. Our support system worked appropriately and was helpful to localize the tumor and determine the resection line.

Image Overlay Support with 3DCG Organ Model for Robot-Assisted Laparoscopic Partial Nephrectomy

We have developed a surgery support system for robot-assisted laparoscopic partial nephrectomy. In our system, a 3D computer graphics (3DCG) model, which includes a kidney, arteries, veins, and tumors, is saved separately and overlaid on the surgeon’s viewing endoscopic camera image through the operator’s console monitor in real time. The position and orientation of the 3DCG model is calculated from the optical flow of the endoscopic camera images, and the model is moved semi-automatically. The display condition of each part of the 3DCG model is independently controlled anytime by using a mouse, rotary controllers, or key board.

Laparoscopic Forceps with Force Feedback

There are two main aspects of safety and effectiveness in laparoscopic surgery. The first is ensuring an appropriate operation field and maintaining this throughout the procedure. The second is finding the correct tissue plane and applying appropriate traction and counter-traction. To accomplish these requirements, surgeons must know the appropriate pressure to apply to the target organ or tissue. For example, weak operation of the forceps in the left hand during laparoscopic surgery leads to poor visibility owing to a small operational field. Furthermore, poor traction at the point of incision on the dissected plane decreases the dissection efficiency. In contrast, when excessive force is applied, there is an increased risk of organ injury or bleeding from capillary vessels during traction; this is clearly detrimental to the overall safety of the procedure. It is difficult for surgeons to master a feeling for the appropriate pressure to apply. Imitating the techniques of skilled surgeons is essential; however, surgical techniques cannot be imitated on first sight. With the aim of assisting this learning process, we developed Forceps Guiding Correct Operation (FOGCOP), new laparoscopic forceps with sensors. Although they are the same shape as Maryland dissecting forceps, FOGCOP can measure the pressure applied on the shaft of the forceps in three axis directions (X, Y, and Z) and on the jaw. The measured pressures are displayed in real time. Surgeons can insert this device into a 5-mm trocar in the same manner as normal forceps. We conducted experiments to verify the effectiveness of the device. 30 students with no experience of laparoscopic surgery participated in this study. Using a training box, students performed a task to press and pull a rubber plate. We compared the performance of a group of students using FOGCOP (group 1, n = 10) with that of a group using conventional forceps (group 2, n = 10). The results suggest that the feedback provided by FOGCOP may be useful for understanding the force delivered by forceps. To verify the usefulness of the device, FOGCOP was also used in laparoscopic nephrectomy of a pig, a procedure that is part of the training course for laparoscopic surgery. It was possible to dissect a tissue plane in the same manner as with conventional forceps. However, the wire from the sensor sometimes interfered with operation. In future, we intend to upgrade FOGCOP by including a wireless sensor, to improve operability to be closer to that of normal forceps, and to allow this device to be used as an educational tool.

Circadian coordination of ATP release in the urothelium via connexin43 hemichannels

Day-night changes in the storage capacity of the urinary bladder are indispensable for sound sleep. Connexin 43 (Cx43), a major gap junction protein, forms hemichannels as a pathway of ATP in other cell types, and the urinary bladder utilizes ATP as a mechanotransduction signals to modulate its capacity. Here, we demonstrate that the circadian clock of the urothelium regulates diurnal ATP release through Cx43 hemichannels. Cx43 was expressed in human and mouse urothelium, and clock genes oscillated in the mouse urothelium accompanied by daily cycles in the expression of Cx43 and extracellular ATP release into the bladder lumen. Equivalent chronological changes in Cx43 and ATP were observed in immortalized human urothelial cells, but these diurnal changes were lost in both arrhythmic Bmal1-knockout mice and in BMAL1-knockdown urothelial cells. ATP release was increased by Cx43 overexpression and was decreased in Cx43 knockdown or in the presence of a selective Cx43 hemichannel blocker, which indicated that Cx43 hemichannels are considered part of the components regulating ATP release in the urothelium. Thus, a functional circadian rhythm exists in the urothelium, and coordinates Cx43 expression and function as hemichannels that provide a direct pathway of ATP release for mechanotransduction and signalling in the urothelium.

PD70-11 CIRCADIAN RHYTHM COORDINATES ATP RELEASE IN THE UROTHELIUM VIA CONNEXIN43 HEMICHANNELS.

INTRODUCTION AND OBJECTIVES: Recent fMRI studies using bladder filling tasks demonstrated alterations in supraspinal LUT control networks (NT) in women with OAB, particularly regarding NT connectivity and white matter changes. It is unclear if patients with OAB show altered supraspinal responses during empty bladder conditions and if resting-state (RS) activity is altered by catheterization usually require for bladder filling tasks. We applied a functionally-motivated NT approach, using RS functional NT connectivity (RS-FNC) analysis, to examine RS related NT interactions in age-matched controls and OAB patients. Based on previous publications we hypothesize lower FNC in OAB patients. METHODS: We examined 10 healthy females (37 9y) and 10 with OAB (38 8y) and DO in urodynamics. For RS-fMRI whole brain images, using a multi-slice EPI sequence were acquired in a 3T scanner. Using SPM8, we estimated the RS NTs using the GIFT toolbox and independent component analysis (ICA) across all subjects. IC dimension estimation was performed using the minimum description length criteria, modified to account for spatial correlation. All nonneuronal ICs (e.g. cardiac-induced pulsatile artifact and head motion) were removed, resulting in a total of 9 neuronal ICs (including the default mode NT, DMN). Prior to FNC analysis, IC time courses were bandpass (0.013 Hz & 0.24 Hz). Group differences in FNC strength were calculated using the FNC toolbox for both conditions. The temporal lags between ICA-derived NTs were computed to gain directed FNC. Significant between-group FNC (and lag) results are shown at p < 0.05 FDR corrected. RESULTS: Healthy controls showed significantly higher (directed) FNC than OAB patients for: DMN (left-dominant) / DMN (right-dominant) and fronto-parietal attention NT / DMN (left-dominant). OAB patients show lower inter-networkcoupling, especially between the DMN(IC1) and the left-dominant fronto-parietal attention NT (FPN,IC4). Further, FNC differed between groups in temporal synchronicity. E.g., lag time between the DMN and FPN is shorter than between the right and left DMN in OAB patients. CONCLUSIONS: We conclude that the interplay between neuronal NTs is altered in OAB patients compared to healthy subjects already during RS. Aberrant coupling of the fronto-parietal attention NT might indicate a general neuronal deficit that impairs adequate LUT control, i.e. suppression of premature micturition reflex, consequently resulting in OAB and incontinence. These novel findings can be an important link to the underlying pathophysiology of OAB in otherwise neurological unimpaired patients.