- Email: [email protected]
Changes in Bladder Wall Blood Oxygen Saturation in the Overactive Obstructed Bladder
Changes in Bladder Wall Blood Oxygen Saturation in the Overactive Obstructed Bladder Jeroen R. Scheepe,* Arjen Amelink, Bas W. D. de Jong, Katja P. Wolffenbuttel and Dirk J. Kok From the Department of Urology and Pediatric Urology and Centre for Optical Diagnostics and Therapy, Department of Radiation Oncology, Erasmus Medical Centre, Sophia Children’s Hospital (AA), Rotterdam, The Netherlands
Abbreviations and Acronyms BOS ⫽ blood oxygen saturation dc ⫽ delivery and collection DPS ⫽ differential path length spectroscopy PAS ⫽ periodic acid-Schiff PBOO ⫽ partial bladder outlet obstruction Pmax ⫽ maximum voiding pressure Wmax ⫽ maximum contractility Submitted for publication November 18, 2010. Study received Erasmus Medical Center animal ethics committee approval. * Correspondence: Department of Urology and Pediatric Urology, Erasmus Medical Center, Sophia Children’s Hospital, P. O. Box 2060, 3000 CB Rotterdam, The Netherlands (telephone: ⫹31-10703 65 59; FAX: ⫹31-10-703 68 02; e-mail: [email protected]).
Purpose: Several studies suggest that hypoxia of the bladder wall contributes to bladder dysfunction but the exact relation between bladder function and blood oxygen saturation, a surrogate marker for hypoxia, is not known. We determined bladder wall blood oxygen saturation in vivo in an animal model of bladder outlet obstruction to establish the exact relation between blood oxygen saturation and bladder function. Materials and Methods: In 8 sham operated and 8 urethrally obstructed guinea pigs we measured blood oxygen saturation of the bladder wall by differential path length spectroscopy before surgery and 8 weeks postoperatively. Urodynamic investigations performed during the whole 8-week period provided data on bladder function. Results: Before surgery and 8 weeks after sham surgery blood oxygen saturation in the bladder wall was between 88% and 95% during filling. It decreased during voiding and returned to greater than 90% within 30 seconds. Eight weeks after obstruction saturation was significantly lower than in the sham operated group during filling and voiding. The decrease was positively related to bladder pressure during filling and voiding, and was more pronounced when overactivity was present. Local bladder contractions occurred without a measurable increase in bladder pressure but were associated with a decrease in saturation. Conclusions: A normal bladder maintains a high oxygen saturation level during filling. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder contractions without a measurable increase in bladder pressure were associated with a decrease in blood saturation. Key Words: urinary bladder; anoxia; urinary bladder, overactive; urethral obstruction; guinea pigs
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THERE is growing evidence that ischemia and reperfusion injury contribute to the initiation of bladder dysfunction.1 Several studies have shown effects in the obstructed bladder that can be interpreted as the long-term results of hypoxia, such as the appearance of glycogen deposits, which is a marker of adaptation to anaerobic metabolism.2– 4
The possible mechanism behind this was already indicated in 1953 in a rat study.5 That study showed that blood flow in the bladder decreases as vessels become compressed by muscle contraction and/or by an intravesical pressure that exceeds intravascular pressure. Later studies confirmed that undistended bladder tissue contains tortuous blood vessels that appear
0022-5347/11/1863-1128/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 1128-1133, September 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.04.111
AND
RESEARCH, INC.
BLOOD OXYGEN SATURATION IN OVERACTIVE OBSTRUCTED BLADDER
ready to unwind upon stretch of the tissue.6,7 It was noted that this decrease in bladder blood flow is accompanied by decreased oxygenation.8,9 This suggests that pressure induced decreases in bladder blood flow are accompanied by decreased blood oxygen saturation, which leads to hypoxic working conditions. To establish the relation between bladder function and hypoxia we performed real-time in vivo measurements of BOS, a surrogate marker for hypoxia, in the bladder wall during complete filling/ voiding cycles in unobstructed and obstructed bladders. BOS was measured twice in individual guinea pigs, that is at the beginning and at the end of a period after obstruction or sham operation. BOS data were related to individual changes in bladder function, which were followed by urodynamics during followup.
MATERIALS AND METHODS Animals and Study Design We used 16 immature male albino Hartley strain guinea pigs. The animals were housed in groups of 4 and allowed a rest period to decrease the stress induced by transport and adjust to the new surroundings. Eight animals with an average weight of 492 gm underwent urethral obstruction and 8 with an average weight of 465 gm underwent sham operation. All 16 animals were followed for 8 weeks. Urodynamic investigations together with DPS were performed at day 0 before surgery in 16 guinea pigs, and 8 weeks after sham operation and obstruction in 8 each that were sacrificed that day.
Experimental Model Surgical procedures and DPS measurement. We used the guinea pig model of PBOO described by Kok10 and Wolffenbuttel11 et al. Obstruction and sham operation were done using ketamine/xylazine anesthesia. The peritoneal cavity was accessed via a lower vertical midline abdominal incision. A silver jeweler jump ring with an internal diameter of 2.2 mm was placed around the bladder neck above the prostate and left there (obstructed group) or removed (sham operated group). A glass fiber probe was then placed directly on the body of the bladder for BOS measurements. At the day of sacrifice a similar midline incision was made to allow probe access to measure BOS of the bladder wall, as described, during multiple filling/voiding cycles. Intravesical pressure was measured simultaneously. The flow rate was not measured during DPS measurements but each DPS measurement sequence was preceded by a complete urodynamic investigation, including flow rate measurement. Urodynamics. Urodynamics were performed at week 0 before obstruction/sham operation and the first DPS measurement, weeks 2, 3, 4, 5, 6 and 7, and week 8 before the second DPS measurement. For each measurement the animals were anesthetized using ketamine (43 mg/kg) and xylazine (0.9 mg/kg) intramuscularly. Through a 24 gauge suprapubic catheter bladder pressure was measured in
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cm H2O and the bladder was filled continuously with sterile saline at a rate of 0.23 ml per minute. The flow rate was measured in ml per second with an ultrasound transducer T106 small animal flow meter (Transonic Systems, Ithaca, New York) around the penis.10 From the urodynamic data we made certain calculations. 1) The number of overactive contractions was defined as a greater than 10 cm H2O increase in intravesical pressure, representing a third of the 95% upper limit of maximum voiding pressure in the unobstructed bladder, without voiding. The number of overactive contractions was considered the number that occurred during 1 filling cycle. We used the average of all cycles during 1 urodynamic study. 2) For Pmax in cm H2O we calculated the average of all voids during 1 urodynamic investigation. 3) Wmax in W/m2 was calculated from the relation between pressure and flow during a voiding according to Griffiths et al.12 The average of all voids during 1 urodynamic investigation was used. 4) The maximal flow rate in ml per second was defined as the highest absolute flow value during voiding. 5) Bladder compliance in ml/cm H2O was defined as the relationship between the change in bladder volume and the change in bladder pressure in the filling phase. Care was taken that pressure values obtained during these periods were not influenced by a nearby voiding or overactive contraction.
Differential Path Length Spectroscopy BOS of the bladder wall was measured in vivo by DPS using glass fibers at the 2 time points when the bladder was accessible. During concomitant bladder pressure measurement the probe was placed in gentle contact with the serosal surface of the anterior bladder wall. To avoid artifacts caused by pressing the probe too hard to the bladder wall the probe was regularly repositioned. The experimental setup used for DPS measurements and the DPS data analysis routine were previously described in detail by Amelink et al.13,14 Briefly, spectra were measured using a fiberoptic probe containing 2, 800 m fibers. Light from a cool white halogen lamp was led through 1 arm of a 200 m bifurcated optical fiber, which was coupled at its distal end to 1 arm (the dc fiber) of the 800 m bifurcated optical fiber probe. The distal end of this fiber probe contacted the tissue under investigation. Light reflected into the dc fiber was coupled back into the 200 m bifurcated fiber and analyzed using a 2-channel spectrometer. Light reflected back from the sample into the other arm of the 800 m bifurcated fiber-optic probe (the collection fiber) was led directly into the second channel of the dual channel spectrometer. The difference in the dc and collection fiber collection signals is called the differential reflectance signal. We previously reported that BOS can be accurately calculated from the differential signal.13,14
Analysis DPS data. Complete sessions consisted of a few hundred to more than a thousand single DPS measurements. During voiding up to 10 DPS measurements and during filling up to hundreds of DPS measurements could be performed. Average saturation was calculated when at least 5 measurement points were available.
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Tissue. After DPS measurement at week 8 the bladders were removed. Half of them were snap frozen and half were fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin for sectioning. Cross-sections (4 m) covering all bladder wall layers were made for staining with the PAS protocol. PAS staining is a routine procedure to visualize sugar moieties that provides specific insight into granular glycogen deposits.3,4 The number of stained granules was counted and scored by 3 researchers in blinded fashion as 0 —absent glycogen granules, 1—a few granules only at the serosal side, 2—light presence throughout the muscle and 3—intense staining up to the urothelium. Previously we noted that this PAS score increases with increasing loss of bladder function in guinea pig and human bladder tissue.4
Before Operation In each group average BOS was between 88% and 95% throughout the filling periods (fig. 3). During voiding average BOS decreased to 85%. The lower limit of individual voids was 62%. BOS returned to around 90% within 30 seconds after voiding. The difference in BOS during voiding compared to BOS during filling was not statistically significant. Week 8 After Sham Operation and Obstruction Eight weeks after sham surgery BOS during filling and voiding was still around 90% (fig. 4). Eight weeks after obstruction average BOS during filling was significantly lower than values at day 0 and values in the sham operated group (p ⬍0.05, fig. 4). Individually BOS decreased to as low as 38%. BOS was also significantly lower during voiding. Individual BOS values during voiding decreased to as low as 12% (fig. 4). To further analyze the filling period in the obstructed group the filling periods were divided into periods with and without overactivity. In the sham operated group this differentiation could not be made since overactivity was almost absent. During filling without overactivity in the obstructed group average ⫾ SD saturation was 93% ⫾ 1%. In the obstructed group BOS was 82% ⫾ 2% in the filling periods without overactivity and 68% ⫾ 5% in the periods with overactivity. The latter value was significantly lower than in the sham operated group (p ⫽ 0.00001, fig. 5). During DPS measurements local bladder contractions during bladder filling were observed occasionally. These local contractions occurred at the site of DPS measurement and at other sites. They appeared to be a real phenomenon that could occur
Statistics. We tested the significance of changes at the 2 time points in the obstructed and sham operated groups with the paired Student t test. Differences between the obstructed and sham operated groups were tested with the unpaired Student t test. Animal experiments were approved by the Erasmus Medical Center animal ethics committee.
RESULTS Urodynamic Data and BOS In the sham operated group compliance, Wmax and Pmax were constant during the whole 8-week period. Overactivity was found only occasionally. Compared to the initial measurement at day 0 and compared to the sham group obstructed animals showed a significant decrease in compliance. and a significant increase in Wmax and Pmax (fig. 1). In all experiments BOS could be measured and correlated to detrusor contractions (fig. 2).
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Figure 1. Urodynamic parameters in sham operated (black bars) and obstructed (white bars) groups. Bars represent average of 8 animals. Asterisk indicates statistically significant (p ⬍0.05).
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Figure 2. BOS (A) in obstructed animal during several complete filling/voiding cycles (B). Saturation decreased during each void. Lowest values occurred several seconds after Pmax. BOS during filling varied between 85% and 100%, and decreased to 40% to 75% during voiding. We noted visible local bladder contractions without concomitant bladder pressure increase. At between 1,300 and 1,400 seconds probe was placed at site of local contractions. During contractions bladder pressure did not change and oxygen saturation at that site decreased. Pves, vesical pressure.
during filling and was not specifically induced by the probe. Local contractions occurred without a measurable increase in bladder pressure but were associated with decreased saturation (fig. 2). In view of the sparse data that we obtained on such situations we could not analyze this further. PAS Staining The average score obtained from PAS staining was 1.0 ⫾ 0.9 in the sham operated group and 2.1 ⫾ 0.9 in the obstructed group (unpaired t test p ⬍0.03).
Figure 3. Percent saturation during filling and voiding in obstructed and sham operated (sham) groups at day 0 before surgery.
DISCUSSION The normal bladder experiences ischemia and reperfusion on a regular basis. During the normal voiding cycle there is a brief period of ischemia at the end of bladder filling as well as during voiding. There is clear evidence that PBOO induces an enhanced decrease in bladder blood flow and hypoxia
Figure 4. Percent saturation during filling and voiding 8 weeks after sham operation (sham) and obstruction. Bars represent lowest, highest and average percent saturation. Vertical lines represent lowest values measured during filling and voiding at week 0 (fig. 3).
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Figure 5. Percent saturation during overactive filling in obstructed animals at week 8 vs filling pressure, calculated as percent saturation ⫽ ⫺0.319 ⫻ filling pressure ⫹ 38.16 (r2 ⫽ 0.83).
in the bladder wall.1 However, there is a lack of information on real-time in vivo measurements of BOS in normal and obstructed bladders. DPS is a noninvasive fiber-optic technique that allows the measurement of hypoxia related parameters in the tissue microvasculature. The reliability of this technique, and its clinical and scientific value have been noted in the oral cavity, bronchial tree, esophagus and breast.14 –17 The deviation between DPS extracted saturation and theoretical saturation of the blood according to the oxygen dissociation curve is less than 3% over the entire 5% to 100% saturation range.18 DPS determines bladder wall BOS as opposed to bladder tissue oxygen tension. Although BOS and tissue oxygen tension may not be similar, they are related.15 Thus, it is reasonable to assume that low BOS correlates with hypoxic working conditions. To our knowledge it is currently unknown whether the microvessels of the 2 bladder wall compartments (urothelium and detrusor) respond identically to bladder pressure changes. With DPS, which features a small interrogation depth of only several hundred m, we only interrogated the serosal side of the bladder wall. No conclusions about the urothelial microvasculature could be drawn from our data. Therefore, as the next step toward understanding the role of the bladder microvasculature in bladder dysfunction, microvascular markers should be measured as a function of filling grade for each compartment separately. Essential in the design of such an experiment is to limit the interrogation depth of the technique to the detrusor microvasculature when measuring from the outside of the bladder wall using a minimally invasive approach, and to the urothelial microvasculature when measuring from the inside of the bladder wall using an endoscopic or catheter based approach. Since the interrogation depth of DPS is proportional to the diameter of the fibers,19 and since DPS probes fit in the working channel of a standard
cystoscope, this can be easily accomplished using DPS with small fiber diameters. We also observed local bladder contractions during bladder filling. The frequency of these bladder movements increased with the appearance of detrusor overactivity (data not shown). Although these spontaneous movements occurred without any measurable increase in bladder pressure, they were associated with decreased BOS (fig. 2). The functional significance of spontaneous activity in the normal bladder during filling is controversial. It was suggested that spontaneous contractions are of neurogenic origin20 and increased bladder sensation is associated with increased local contractile activity in the bladder wall.21 However, our data suggest a correlation between local bladder contractions and bladder wall ischemia. Thus, local contractile activity in the bladder wall may represent a mechanism that adds to the pathophysiology of bladder dysfunction that is not revealed by urodynamics alone. In our experimental setup we measured the BOS in the detrusor muscle. As our data show, a normally functioning detrusor muscle maintains high BOS. PBOO compromises this level of saturation during filling, especially when overactivity is involved. These findings were supported by increased glycogen, a tissue marker for hypoxia, in the bladder wall of obstructed animals.3,4 Furthermore, the decrease in BOS was more pronounced during overactive filling at high filling pressure. This could represent a self-enhancing cycle in which each high pressure/ overactive period induces ischemia, which affects bladder muscle and nerve cells, inducing new overactivity and a new ischemia/reperfusion cycle. This would greatly enhance the total time that the tissue experiences ischemia from only a few minutes daily during voiding for the healthy bladder to extended periods during filling for the overactive/obstructed bladder. There is evidence that oxidative stress is a key feature in the initiation and progression of voiding dysfunction.1 Normalizing bladder oxidative stress might reverse bladder dysfunction after ischemia/reperfusion injury. These data were obtained from animal experimental studies. Since DPS probes fit in the working channel of a standard cystoscope, this technique can be used in humans and animals to monitor how BOS changes due to bladder dysfunction with or without PBOO and during interventions aimed specifically at bladder function and/or improving bladder blood circulation. There is some evidence that treatment with anticholinergics might positively influence bladder wall blood oxygen saturation,22 which can be measured by DPS. Furthermore, it is interesting to study possible correlations between bladder wall oxygenation and bladder function deterioration/rehabilitation. This could be done in an animal model, eg by
BLOOD OXYGEN SATURATION IN OVERACTIVE OBSTRUCTED BLADDER
measuring BOS and bladder function in our guinea pig model before obstruction, during de-obstruction and after a recovery period.
CONCLUSIONS A normally functioning bladder maintains high blood oxygen saturation level during filling and voiding. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder
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contractions without a measurable increase in bladder pressure were associated with decreased blood saturation, which suggests a possible role in the pathophysiology of overactive bladder. Bladder blood oxygen saturation can be monitored noninvasively, which can have clinical and scientific value. DPS is a promising technique for future clinical and experimental studies in which quantitative, accurate measurement of bladder wall blood oxygen saturation is indicated.
REFERENCES 1. Brading A, Pessina F, Esposito L et al: Effects of metabolic stress and ischaemia on the bladder, and the relationship with bladder overactivity. Scand J Urol Nephrol Suppl 2004; 215: 84.
8. Greenland JE and Brading AF: The effect of bladder outflow obstruction on detrusor blood flow changes during the voiding cycle in conscious pigs. J Urol 2001; 165: 245.
2. Pessina F, Solito R, Maestrini D et al: Effect of anoxia-glucopenia and re-superfusion on intrinsic nerves of mammalian detrusor smooth muscle: importance of glucose metabolism. Neurourol Urodyn 2005; 24: 389.
9. Azadzoi KM, Pontari M, Vlachiotis J et al: Canine bladder blood flow and oxygenation: changes induced by filling, contraction and outlet obstruction. J Urol 1996; 155: 1459.
3. de Jong BWD, Bakker Schut TC, Coppens J et al: Raman spectroscopic detection of changes in molecular composition of bladder muscle tissue caused by outlet obstruction. Vibrat Spectrosc 2003; 32: 57. 4. de Jong BW, Wolffenbuttel KP, Scheepe JR et al: The detrusor glycogen content of a de-obstructed bladder reflects the functional history of that bladder during PBOO. Neurourol Urodyn 2008; 27: 454.
10. Kok DJ, Wolffenbuttel KP, Minekus JP et al: Changes in bladder contractility and compliance due to urethral obstruction: a longitudinal followup of guinea pigs. J Urol 2000; 164: 1021. 11. Wolffenbuttel KP, Kok DJ, Minekus JP et al: Urodynamic follow-up of experimental urethral obstruction in individual guinea pigs. Neurourol Urodyn 2001; 20: 699.
using optical spectroscopy. Lung Cancer 2007; 57: 317. 16. Van Veen RLP, Amelink A, Menke-Pluymers M et al: Optical biopsy of breast tissue using differential path-length spectroscopy. Phys Med Biol 2005; 50: 2573. 17. Amelink A, Haringsma J and Sterenborg HJC: Noninvasive measurement of oxygen saturation of the microvascular blood in Barrett’s dysplasia by use of optical spectroscopy. Gastrointest Endosc 2009; 70: 1. 18. Amelink A, Christiaanse T and Sterenborg HJCM: Effect of hemoglobin extinction spectra on optical spectroscopic measurements of blood oxygen saturation. Opt Lett 2009; 34: 1525.
12. Griffiths DJ, Constantinou CE and van Mastrigt R: Urinary bladder function and its control in normal females. Am J Physiol 1986; 251: R225.
19. Kaspers OP, Sterenborg HJCM and Amelink A: Controlling the optical path length in turbid media using differential path-length spectroscopy: fiber diameter dependence. Appl Optics 2008; 47: 365.
5. Mehrotra RML: An experimental study of the vesical circulation during distension and in cystitis. J Pathol Bacteriol 1953; 66: 79.
13. Amelink A and Sterenborg HJCM: Measurement of the local optical properties of turbid media by differential path-length spectroscopy. Appl Optics 2004; 43: 3048.
20. Levin RM, Ruggieri MR, Velagapudi S et al: Relevance of spontaneous activity to urinary bladder function: an in vitro and in vivo study. J Urol 1986; 136: 517.
6. Hossler FE and Kao RL: Microvasculature of the urinary bladder of the dog: a study using vascular corrosion casting. Microsc Micoanal 2007; 13: 220.
14. Amelink A, Kaspers OP, Sterenborg HJCM et al: Non-invasive measurement of the morphology and physiology of oral mucosa by use of optical spectroscopy. Oral Oncol 2008; 44: 65.
21. Drake MJ, Harvey IJ, Gillespi JI et al: Localized contractions in the normal human bladder and in urinary urgency. BJU Int 2005; 95: 1002.
7. Miodonski A and Litwin J: Microvascular architecture of the human urinary bladder wall: a corrosion casting study. Anat Rec 1999; 254: 375.
15. Aerts JGJV, Amelink A, Van der Leest C et al: HIF1a expression in bronchial biopsies correlates with tumor microvascular saturation determined
22. Scheepe JR, de Jong BW, Wolffenbuttel KP et al: The effect of oxybutynin on structural changes of the obstructed guinea pig bladder. J Urol 2007; 178: 1807.
Abbreviations and Acronyms BOS ⫽ blood oxygen saturation dc ⫽ delivery and collection DPS ⫽ differential path length spectroscopy PAS ⫽ periodic acid-Schiff PBOO ⫽ partial bladder outlet obstruction Pmax ⫽ maximum voiding pressure Wmax ⫽ maximum contractility Submitted for publication November 18, 2010. Study received Erasmus Medical Center animal ethics committee approval. * Correspondence: Department of Urology and Pediatric Urology, Erasmus Medical Center, Sophia Children’s Hospital, P. O. Box 2060, 3000 CB Rotterdam, The Netherlands (telephone: ⫹31-10703 65 59; FAX: ⫹31-10-703 68 02; e-mail: [email protected]).
Purpose: Several studies suggest that hypoxia of the bladder wall contributes to bladder dysfunction but the exact relation between bladder function and blood oxygen saturation, a surrogate marker for hypoxia, is not known. We determined bladder wall blood oxygen saturation in vivo in an animal model of bladder outlet obstruction to establish the exact relation between blood oxygen saturation and bladder function. Materials and Methods: In 8 sham operated and 8 urethrally obstructed guinea pigs we measured blood oxygen saturation of the bladder wall by differential path length spectroscopy before surgery and 8 weeks postoperatively. Urodynamic investigations performed during the whole 8-week period provided data on bladder function. Results: Before surgery and 8 weeks after sham surgery blood oxygen saturation in the bladder wall was between 88% and 95% during filling. It decreased during voiding and returned to greater than 90% within 30 seconds. Eight weeks after obstruction saturation was significantly lower than in the sham operated group during filling and voiding. The decrease was positively related to bladder pressure during filling and voiding, and was more pronounced when overactivity was present. Local bladder contractions occurred without a measurable increase in bladder pressure but were associated with a decrease in saturation. Conclusions: A normal bladder maintains a high oxygen saturation level during filling. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder contractions without a measurable increase in bladder pressure were associated with a decrease in blood saturation. Key Words: urinary bladder; anoxia; urinary bladder, overactive; urethral obstruction; guinea pigs
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THERE is growing evidence that ischemia and reperfusion injury contribute to the initiation of bladder dysfunction.1 Several studies have shown effects in the obstructed bladder that can be interpreted as the long-term results of hypoxia, such as the appearance of glycogen deposits, which is a marker of adaptation to anaerobic metabolism.2– 4
The possible mechanism behind this was already indicated in 1953 in a rat study.5 That study showed that blood flow in the bladder decreases as vessels become compressed by muscle contraction and/or by an intravesical pressure that exceeds intravascular pressure. Later studies confirmed that undistended bladder tissue contains tortuous blood vessels that appear
0022-5347/11/1863-1128/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 1128-1133, September 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.04.111
AND
RESEARCH, INC.
BLOOD OXYGEN SATURATION IN OVERACTIVE OBSTRUCTED BLADDER
ready to unwind upon stretch of the tissue.6,7 It was noted that this decrease in bladder blood flow is accompanied by decreased oxygenation.8,9 This suggests that pressure induced decreases in bladder blood flow are accompanied by decreased blood oxygen saturation, which leads to hypoxic working conditions. To establish the relation between bladder function and hypoxia we performed real-time in vivo measurements of BOS, a surrogate marker for hypoxia, in the bladder wall during complete filling/ voiding cycles in unobstructed and obstructed bladders. BOS was measured twice in individual guinea pigs, that is at the beginning and at the end of a period after obstruction or sham operation. BOS data were related to individual changes in bladder function, which were followed by urodynamics during followup.
MATERIALS AND METHODS Animals and Study Design We used 16 immature male albino Hartley strain guinea pigs. The animals were housed in groups of 4 and allowed a rest period to decrease the stress induced by transport and adjust to the new surroundings. Eight animals with an average weight of 492 gm underwent urethral obstruction and 8 with an average weight of 465 gm underwent sham operation. All 16 animals were followed for 8 weeks. Urodynamic investigations together with DPS were performed at day 0 before surgery in 16 guinea pigs, and 8 weeks after sham operation and obstruction in 8 each that were sacrificed that day.
Experimental Model Surgical procedures and DPS measurement. We used the guinea pig model of PBOO described by Kok10 and Wolffenbuttel11 et al. Obstruction and sham operation were done using ketamine/xylazine anesthesia. The peritoneal cavity was accessed via a lower vertical midline abdominal incision. A silver jeweler jump ring with an internal diameter of 2.2 mm was placed around the bladder neck above the prostate and left there (obstructed group) or removed (sham operated group). A glass fiber probe was then placed directly on the body of the bladder for BOS measurements. At the day of sacrifice a similar midline incision was made to allow probe access to measure BOS of the bladder wall, as described, during multiple filling/voiding cycles. Intravesical pressure was measured simultaneously. The flow rate was not measured during DPS measurements but each DPS measurement sequence was preceded by a complete urodynamic investigation, including flow rate measurement. Urodynamics. Urodynamics were performed at week 0 before obstruction/sham operation and the first DPS measurement, weeks 2, 3, 4, 5, 6 and 7, and week 8 before the second DPS measurement. For each measurement the animals were anesthetized using ketamine (43 mg/kg) and xylazine (0.9 mg/kg) intramuscularly. Through a 24 gauge suprapubic catheter bladder pressure was measured in
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cm H2O and the bladder was filled continuously with sterile saline at a rate of 0.23 ml per minute. The flow rate was measured in ml per second with an ultrasound transducer T106 small animal flow meter (Transonic Systems, Ithaca, New York) around the penis.10 From the urodynamic data we made certain calculations. 1) The number of overactive contractions was defined as a greater than 10 cm H2O increase in intravesical pressure, representing a third of the 95% upper limit of maximum voiding pressure in the unobstructed bladder, without voiding. The number of overactive contractions was considered the number that occurred during 1 filling cycle. We used the average of all cycles during 1 urodynamic study. 2) For Pmax in cm H2O we calculated the average of all voids during 1 urodynamic investigation. 3) Wmax in W/m2 was calculated from the relation between pressure and flow during a voiding according to Griffiths et al.12 The average of all voids during 1 urodynamic investigation was used. 4) The maximal flow rate in ml per second was defined as the highest absolute flow value during voiding. 5) Bladder compliance in ml/cm H2O was defined as the relationship between the change in bladder volume and the change in bladder pressure in the filling phase. Care was taken that pressure values obtained during these periods were not influenced by a nearby voiding or overactive contraction.
Differential Path Length Spectroscopy BOS of the bladder wall was measured in vivo by DPS using glass fibers at the 2 time points when the bladder was accessible. During concomitant bladder pressure measurement the probe was placed in gentle contact with the serosal surface of the anterior bladder wall. To avoid artifacts caused by pressing the probe too hard to the bladder wall the probe was regularly repositioned. The experimental setup used for DPS measurements and the DPS data analysis routine were previously described in detail by Amelink et al.13,14 Briefly, spectra were measured using a fiberoptic probe containing 2, 800 m fibers. Light from a cool white halogen lamp was led through 1 arm of a 200 m bifurcated optical fiber, which was coupled at its distal end to 1 arm (the dc fiber) of the 800 m bifurcated optical fiber probe. The distal end of this fiber probe contacted the tissue under investigation. Light reflected into the dc fiber was coupled back into the 200 m bifurcated fiber and analyzed using a 2-channel spectrometer. Light reflected back from the sample into the other arm of the 800 m bifurcated fiber-optic probe (the collection fiber) was led directly into the second channel of the dual channel spectrometer. The difference in the dc and collection fiber collection signals is called the differential reflectance signal. We previously reported that BOS can be accurately calculated from the differential signal.13,14
Analysis DPS data. Complete sessions consisted of a few hundred to more than a thousand single DPS measurements. During voiding up to 10 DPS measurements and during filling up to hundreds of DPS measurements could be performed. Average saturation was calculated when at least 5 measurement points were available.
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Tissue. After DPS measurement at week 8 the bladders were removed. Half of them were snap frozen and half were fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin for sectioning. Cross-sections (4 m) covering all bladder wall layers were made for staining with the PAS protocol. PAS staining is a routine procedure to visualize sugar moieties that provides specific insight into granular glycogen deposits.3,4 The number of stained granules was counted and scored by 3 researchers in blinded fashion as 0 —absent glycogen granules, 1—a few granules only at the serosal side, 2—light presence throughout the muscle and 3—intense staining up to the urothelium. Previously we noted that this PAS score increases with increasing loss of bladder function in guinea pig and human bladder tissue.4
Before Operation In each group average BOS was between 88% and 95% throughout the filling periods (fig. 3). During voiding average BOS decreased to 85%. The lower limit of individual voids was 62%. BOS returned to around 90% within 30 seconds after voiding. The difference in BOS during voiding compared to BOS during filling was not statistically significant. Week 8 After Sham Operation and Obstruction Eight weeks after sham surgery BOS during filling and voiding was still around 90% (fig. 4). Eight weeks after obstruction average BOS during filling was significantly lower than values at day 0 and values in the sham operated group (p ⬍0.05, fig. 4). Individually BOS decreased to as low as 38%. BOS was also significantly lower during voiding. Individual BOS values during voiding decreased to as low as 12% (fig. 4). To further analyze the filling period in the obstructed group the filling periods were divided into periods with and without overactivity. In the sham operated group this differentiation could not be made since overactivity was almost absent. During filling without overactivity in the obstructed group average ⫾ SD saturation was 93% ⫾ 1%. In the obstructed group BOS was 82% ⫾ 2% in the filling periods without overactivity and 68% ⫾ 5% in the periods with overactivity. The latter value was significantly lower than in the sham operated group (p ⫽ 0.00001, fig. 5). During DPS measurements local bladder contractions during bladder filling were observed occasionally. These local contractions occurred at the site of DPS measurement and at other sites. They appeared to be a real phenomenon that could occur
Statistics. We tested the significance of changes at the 2 time points in the obstructed and sham operated groups with the paired Student t test. Differences between the obstructed and sham operated groups were tested with the unpaired Student t test. Animal experiments were approved by the Erasmus Medical Center animal ethics committee.
RESULTS Urodynamic Data and BOS In the sham operated group compliance, Wmax and Pmax were constant during the whole 8-week period. Overactivity was found only occasionally. Compared to the initial measurement at day 0 and compared to the sham group obstructed animals showed a significant decrease in compliance. and a significant increase in Wmax and Pmax (fig. 1). In all experiments BOS could be measured and correlated to detrusor contractions (fig. 2).
Maximum voiding pressure 80
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Figure 1. Urodynamic parameters in sham operated (black bars) and obstructed (white bars) groups. Bars represent average of 8 animals. Asterisk indicates statistically significant (p ⬍0.05).
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Figure 2. BOS (A) in obstructed animal during several complete filling/voiding cycles (B). Saturation decreased during each void. Lowest values occurred several seconds after Pmax. BOS during filling varied between 85% and 100%, and decreased to 40% to 75% during voiding. We noted visible local bladder contractions without concomitant bladder pressure increase. At between 1,300 and 1,400 seconds probe was placed at site of local contractions. During contractions bladder pressure did not change and oxygen saturation at that site decreased. Pves, vesical pressure.
during filling and was not specifically induced by the probe. Local contractions occurred without a measurable increase in bladder pressure but were associated with decreased saturation (fig. 2). In view of the sparse data that we obtained on such situations we could not analyze this further. PAS Staining The average score obtained from PAS staining was 1.0 ⫾ 0.9 in the sham operated group and 2.1 ⫾ 0.9 in the obstructed group (unpaired t test p ⬍0.03).
Figure 3. Percent saturation during filling and voiding in obstructed and sham operated (sham) groups at day 0 before surgery.
DISCUSSION The normal bladder experiences ischemia and reperfusion on a regular basis. During the normal voiding cycle there is a brief period of ischemia at the end of bladder filling as well as during voiding. There is clear evidence that PBOO induces an enhanced decrease in bladder blood flow and hypoxia
Figure 4. Percent saturation during filling and voiding 8 weeks after sham operation (sham) and obstruction. Bars represent lowest, highest and average percent saturation. Vertical lines represent lowest values measured during filling and voiding at week 0 (fig. 3).
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BLOOD OXYGEN SATURATION IN OVERACTIVE OBSTRUCTED BLADDER
Figure 5. Percent saturation during overactive filling in obstructed animals at week 8 vs filling pressure, calculated as percent saturation ⫽ ⫺0.319 ⫻ filling pressure ⫹ 38.16 (r2 ⫽ 0.83).
in the bladder wall.1 However, there is a lack of information on real-time in vivo measurements of BOS in normal and obstructed bladders. DPS is a noninvasive fiber-optic technique that allows the measurement of hypoxia related parameters in the tissue microvasculature. The reliability of this technique, and its clinical and scientific value have been noted in the oral cavity, bronchial tree, esophagus and breast.14 –17 The deviation between DPS extracted saturation and theoretical saturation of the blood according to the oxygen dissociation curve is less than 3% over the entire 5% to 100% saturation range.18 DPS determines bladder wall BOS as opposed to bladder tissue oxygen tension. Although BOS and tissue oxygen tension may not be similar, they are related.15 Thus, it is reasonable to assume that low BOS correlates with hypoxic working conditions. To our knowledge it is currently unknown whether the microvessels of the 2 bladder wall compartments (urothelium and detrusor) respond identically to bladder pressure changes. With DPS, which features a small interrogation depth of only several hundred m, we only interrogated the serosal side of the bladder wall. No conclusions about the urothelial microvasculature could be drawn from our data. Therefore, as the next step toward understanding the role of the bladder microvasculature in bladder dysfunction, microvascular markers should be measured as a function of filling grade for each compartment separately. Essential in the design of such an experiment is to limit the interrogation depth of the technique to the detrusor microvasculature when measuring from the outside of the bladder wall using a minimally invasive approach, and to the urothelial microvasculature when measuring from the inside of the bladder wall using an endoscopic or catheter based approach. Since the interrogation depth of DPS is proportional to the diameter of the fibers,19 and since DPS probes fit in the working channel of a standard
cystoscope, this can be easily accomplished using DPS with small fiber diameters. We also observed local bladder contractions during bladder filling. The frequency of these bladder movements increased with the appearance of detrusor overactivity (data not shown). Although these spontaneous movements occurred without any measurable increase in bladder pressure, they were associated with decreased BOS (fig. 2). The functional significance of spontaneous activity in the normal bladder during filling is controversial. It was suggested that spontaneous contractions are of neurogenic origin20 and increased bladder sensation is associated with increased local contractile activity in the bladder wall.21 However, our data suggest a correlation between local bladder contractions and bladder wall ischemia. Thus, local contractile activity in the bladder wall may represent a mechanism that adds to the pathophysiology of bladder dysfunction that is not revealed by urodynamics alone. In our experimental setup we measured the BOS in the detrusor muscle. As our data show, a normally functioning detrusor muscle maintains high BOS. PBOO compromises this level of saturation during filling, especially when overactivity is involved. These findings were supported by increased glycogen, a tissue marker for hypoxia, in the bladder wall of obstructed animals.3,4 Furthermore, the decrease in BOS was more pronounced during overactive filling at high filling pressure. This could represent a self-enhancing cycle in which each high pressure/ overactive period induces ischemia, which affects bladder muscle and nerve cells, inducing new overactivity and a new ischemia/reperfusion cycle. This would greatly enhance the total time that the tissue experiences ischemia from only a few minutes daily during voiding for the healthy bladder to extended periods during filling for the overactive/obstructed bladder. There is evidence that oxidative stress is a key feature in the initiation and progression of voiding dysfunction.1 Normalizing bladder oxidative stress might reverse bladder dysfunction after ischemia/reperfusion injury. These data were obtained from animal experimental studies. Since DPS probes fit in the working channel of a standard cystoscope, this technique can be used in humans and animals to monitor how BOS changes due to bladder dysfunction with or without PBOO and during interventions aimed specifically at bladder function and/or improving bladder blood circulation. There is some evidence that treatment with anticholinergics might positively influence bladder wall blood oxygen saturation,22 which can be measured by DPS. Furthermore, it is interesting to study possible correlations between bladder wall oxygenation and bladder function deterioration/rehabilitation. This could be done in an animal model, eg by
BLOOD OXYGEN SATURATION IN OVERACTIVE OBSTRUCTED BLADDER
measuring BOS and bladder function in our guinea pig model before obstruction, during de-obstruction and after a recovery period.
CONCLUSIONS A normally functioning bladder maintains high blood oxygen saturation level during filling and voiding. Bladder obstruction compromises this ability, especially when it involves overactivity. Local bladder
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contractions without a measurable increase in bladder pressure were associated with decreased blood saturation, which suggests a possible role in the pathophysiology of overactive bladder. Bladder blood oxygen saturation can be monitored noninvasively, which can have clinical and scientific value. DPS is a promising technique for future clinical and experimental studies in which quantitative, accurate measurement of bladder wall blood oxygen saturation is indicated.
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