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MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF HUMAN DETRUSOR MITOCHONDRIA WITH URODYNAMIC CORRELATION AFTER PARTIAL BLADDER OUTLET OBSTRUCTION
0022-5347/00/1631-0225/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 163, 225–229, January 2000 Printed in U.S.A.
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF HUMAN DETRUSOR MITOCHONDRIA WITH URODYNAMIC CORRELATION AFTER PARTIAL BLADDER OUTLET OBSTRUCTION SHING-HWA LU,* YAU-HUEI WEI, LUKE S. CHANG, ALEX T. L. LIN, KUANG-KUO CHEN AND AN-HANG YANG†,‡ From the Divisions of Urology (Department of Surgery) and Ultrastructural and Molecular Pathology (Department of Pathology), Veterans General Hospital-Taipei Institute of Clinical Medicine, School of Medicine and Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China
ABSTRACT
Purpose: We correlated ultrastructural changes in mitochondria in the human detrusor with the severity of partial bladder outlet obstruction on urodynamics. Materials and Methods: We recruited into the study 52 men with and without bladder outlet obstruction symptoms. The severity of partial bladder outlet obstruction was determined by pressure flow study. Random detrusor biopsy specimens obtained by cystoscopy were fixed immediately and processed for transmission electron microscopic observation. Random areas were photographed for further morphological and morphometric analysis using mitochondrial damage score and stereological principles. Results: Mitochondrial damage score and mean mitochondrial volume strongly correlated with the urodynamic severity of partial bladder outlet obstruction, while mitochondrial volume density, surface density of the mitochondrial outer membrane and number of mitochondria per unit of cytoplasm area did not significantly correlate with severity. Conclusions: Detrusor mitochondrial swelling and structural destruction increased with the severity of partial bladder outlet obstruction. These changes may be associated with impaired mitochondrial function and oxidative metabolism after partial bladder outlet obstruction. Detrusor mitochondrial damage may explain voiding dysfunction after partial bladder outlet obstruction develops. KEY WORDS: bladder, urodynamics, mitochondria, bladder neck obstruction
Bladder outlet obstruction causes significant morbidity, including irritative voiding symptoms, voiding difficulty and even urinary retention with or without detrusor failure. However, the pathogenesis of these disorders has not been fully elucidated. Some ultrastructural studies have revealed widely separated muscle cells with intermediate cell junction reduction, collagenosis and hypertrophic muscle cells in human and animal bladders after the development of bladder outlet obstruction.1, 2 Elbadawi et al also observed swollen, disrupted and vacuolated detrusor mitochondria in partial bladder outlet obstruction.1, 2 Recently we reported that impaired mitochondrial enzyme function has an important role in decreasing energy production and contractility of the detrusor in bladder outlet obstruction in rabbits.3 Furthermore, some groups have performed morphometry of the mitochondria in various tissues other than the detrusor in human and animals under different physiological or pathological conditions.4 – 6 However, to our knowledge there has been no quantitative study of human detrusor mitochondria in partial bladder outlet obstruction. We used stereological principles and mitochondrial damage score to evaluate morphological changes in detrusor mitochondria in various grades of partial bladder outlet obstruction severity, as determined by urodynamics.
MATERIALS AND METHODS
Patients and specimen processing. From December 1996 to November 1998, 67 men with and without obstructive voiding symptoms were evaluated in this study. Those without obstructive voiding symptoms had a ureteral stone, stricture or tumor. Partial obstruction was defined as bladder outlet obstruction that was not completely occlusive. Excluded from study were 15 patients with possbile neurogenic bladder, including those with stroke, spinal cord disease, diabetes mellitus, Parkinsonism and spinal bifida. A total of 52 men 34 to 79 years old (mean age 68.2) were enrolled in this study, and evaluated by the International Prostate Symptom Score (I-PSS) and quality of life assessment index (table 1). Written informed consent was obtained from each patient. Preoperatively 5 cold cup detrusor biopsies were performed randomly in each lateral bladder wall using spinal anesthesia. Each biopsy specimen was less than 1 mm.3. Specimens were immediately fixed with 2.5% glutaraldehyde in phosphate buffer, pH 7.4, for 2 hours at room temperature. They were thoroughly washed, post-fixed with 1% buffered osmium tetroxide, and subsequently processed with standard dehydration and embedding procedures. Ultrathin sections (70 nm.) were doubly stained with uranyl acetate and lead citrate,7 and examined with a transmission electron microscope at 60 kV. Urodynamic studies (table 1). Medication, such as antihistamine, that would interfere with detrusor function was discontinued for at least 3 weeks before urodynamics. In addition, patients with a urinary tract infection were excluded from study. Medium fill (30 ml. per minute) cystometry with a pressure flow study was performed at least 2 days before detrusor biopsy in each case. Cystometry was done by infus-
Accepted for publication August 13, 1999. * Recipient of Veterans General Hospital-Taipei and National Yang-Ming University Research Grants VGH-262, 1998 and VGHYM87-S1 to 08 1998. † Recipient of Veterans General Hospital-Taipei and National Yong-Ming University Grant VGH-360, 1998. ‡ Requests for reprints: Division of Ultrastructural and Molecular Pathology, Department of Pathology, Veterans General Hospital– Taipei, No. 201, Section 2, Shih Pai Rd., Taipei 112, Taiwan, Republic of China. 225
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TABLE 1. Clinical characteristics of patients with obstruction of various grades as defined by the linearized passive urethral resistance relation nomogram Mean 6 SD (range) 0 No. pts. Age Yrs. symptoms I-PSS (range 0–35) Quality of life assessment index (range 0–6) PSA (ng./ml.) Prostate size (ml.) Max. flow rate (ml./sec.) Vol. at initial urge (ml.) Bladder capacity (ml.) Post-void residual urine (ml.)
11 55.1 6 9.75 0.27 6 0.61 5.36 6 3.38 0.73 6 0.65
1 (34.0) (2.00) (12.0) (2.00)
11 72.7 6 4.31 3.86 6 2.81 14.5 6 5.50 3.64 6 1.03
(13.0) (9.00) (17.0) (3.00)
2
3
4
6 72.5 6 3.39 (9.00) 3.47 6 2.58 (6.80) 16.3 6 9.42 (23.0) 4.17 6 0.75 (2.00)
12 70.5 6 9.14 (33.0) 3.96 6 2.67 (9.00) 17.2 6 5.97 (17.0) 3.92 6 1.08 (3.00)
9 71.7 6 4.85 (14.0) 2.78 6 2.03 (5.00) 21.0 6 3.64 (13.0) 4.67 6 0.71 (2.00)
5 3 71.7 6 6.81 3.67 6 0.76 17.3 6 1.53 4.67 6 0.58
(13.0) (1.50) (3.00) (1.00)
1.21 6 0.74 (2.28) 3.45 6 2.60 (7.84) 4.07 6 2.18 (5.50) 3.48 6 1.26 (4.57) 3.34 6 1.90 (5.24) 4.91 6 2.59 (4.56) 24.1 6 3.75 (15.0) 36.1 6 20.5 (71.3) 39.3 6 15.1 (32.3) 37.2 6 22.2 (70.3) 57.3 6 25.9 (84.4) 41.1 6 18.7 (34.7) 20.1 6 4.81 (14.0) 9.00 6 2.57 (10.0) 7.00 6 2.76 (6.00) 8.75 6 2.56 (8.00) 7.89 6 2.32 (6.00) 9.00 6 2.65 (5.00) 253.1 6 75.2 (269) 152.5 6 96.3 (352) 200.0 6 87.9 (207) 156.7 6 47.3 (146) 123.8 6 67.9 (178) 164.3 6 144.7 (267) 397.6 6 125.9 (432) 197.0 6 107.2 (357) 264.5 6 89.3 (222) 231.3 6 69.7 (202) 184.6 6 86.4 (203) 244.7 6 186.1 (364) 8.18 6 7.51 (20.0) 21.0 6 12.2 (40.0) 16.7 6 12.5 (35.0) 53.3 6 79.4 (290) 100.0 6 73.0 (235) 116.7 6 76.4 (150)
ing sterile water at room temperature through an 8Fr urethral catheter. A 5Fr urethral catheter and rectal catheter were inserted and connected to fluid filled transducers at the level of the symphysis pubis to measure intravesical and abdominal pressure, respectively. Detrusor pressure was determined by subtracting abdominal from intravesical pressure. Bladder compliance was not significantly decreased. Subsequently we plotted the pressure flow study data on the linearized passive urethral resistance relation and International Continence Society nomograms8, 9 with patients classified into groups according to the severity of partial bladder outlet obstruction. All evaluations were performed by one of us (S. H. L.). For transmission electron microscopic observation 6 photographs of different fields were obtained randomly in each random biopsy specimen at 33,176 magnification. For each micrograph the whole area was reviewed and all mitochondria were evaluated. Magnification of all micrographs was calibrated using 2 carbon grating replicas. For mitochondrial score each negative at 33,176 magnification was examined under a dissecting microscopy at 3110 magnification by one of us (S. H. L.). Mitochondrial damage score was assessed blindly using the methods described by Flameng10 and Schirmer et al.11 Each mitochondrion was scored as grade 0 to 4 to represent the severity of structural destruction. Mean scores of all mitochondria (approximately 500) per case were then calculated. Morphometric analysis. Detrusor mitochondria were analyzed quantitatively by point counting.12, 13 Negatives were projected by an enlarger onto a screen covered with a sheet of square lattice of points at 3 mm. intervals and a sheet of test lines 3 mm. long with 2.6 mm. between 2 parallel test lines. Intersections of the square lattice lines or the end points of the test line were considered test points. Test points or test lines over the mitochondria, cytoplasm and outer mitochondrial surface were counted and totaled in each photograph. According to previously described stereological and morphometric principles, 6, 12, 13 certain formulas were used to estimate morphometric parameters. Mean mitochondrial volume, used to evaluate the size of individual mitochondria, was estimated by dividing the area or number of test points falling on the mitochondria (Pi) by the total number of mitochondria (Nt). Mitochondria volume density or volume fraction per unit volume of cytoplasm (Vv) was measured as Pi/Pc, where Pc represents the number of test points falling on the cytoplasm. Using the test line system surface density (Sv) of the mitochondrial outer membrane was estimated using the formula, Sv 5 4 3 Ii/Pc 3 LT, where Sv represents the mitochondrial outer membrane surface fraction per unit volume of cytoplasm, Ii the number of test line intersections with mitochondrial outer membrane and LT the test line length. The number of mitochondria per unit of cytoplasm area (NA) was calculated as Nt/Pc.
Statistics. We calculated the mean value plus or minus standard error and 95% confidence interval of the morphometric analysis and mitochondrial scores in each case. To evaluate the association of clinical data with transmission electron microscope findings we performed multiple linear regression and 1-way analysis of variance by the Tukey method for post hoc multiple comparisons using computer software with p ,0.05 considered statistically significant. RESULTS
According to the linearized passive urethral resistance relation nomogram 11 cases were categorized as grade 0, 11–grade 1, 6–grade 2, 12–grade 3, 9–grade 4 and 3–grade 5. Table 1 shows the urodynamics data. Transmission electron microscopy revealed progressive detrusor mitochondrial changes, including a loss of granules, swelling, condensation and clarification of the mitochondrial matrix, and disruption of the inner and outer membranes as the severity of partial bladder outlet obstruction increased (fig. 1). Mean mitochondrial score correlated well with the severity of bladder outlet obstruction, as determined by linearized passive urethral resistance relation obstructive grade8 (fig. 2). When patients were divided into nonobstructive, equivocal and obstructive groups according to the International Continence Society nomogram,9 mitochondrial score showed a statistically significant difference among the 3 groups (p ,0.001). Moreover, comparing any 2 groups revealed significant differences (p ,0.01, table 2). In addition, the mitochondrial score in each individual mitochondrion appeared different in the same cell of the detrusor. Mean mitochondrial score was higher in the 24 patients with than the 28 without detrusor instability (1.543 6 0.090 versus 1.066 6 0.136, p 5 0.007). Morphometric analysis revealed that the mean volume of mitochondria positively correlated with the severity of partial bladder outlet obstruction, as determined by linearized passive urethral resistance relation obstructive grade (fig. 3). Mitochondria volume density, surface density of the mitochondrial outer membrane and number of mitochondria per unit of cytoplasm area did not correlate significantly with the severity of partial bladder outlet obstruction, as classified by obstructive grade (figs. 4 to 6). When patients were divided into nonobstructive, equivocal and obstructive groups according to the International Continence Society nomogram, only mean mitochondrial volume revealed a statistically significant difference (p 5 0.015) among the 3 groups. Furthermore, when any 2 groups were compared, mean mitochondrial volume in the nonobstructive group was smaller than that in the equivocal or obstructive group (p ,0.05). However, the difference in the equivocal and obstructive groups was not statistically significant (p 5 0.917, table 2). Mitochondria volume density, surface density of the mitochondrial outer membrane and number of mito-
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF DETRUSOR MITOCHONDRIA
227
FIG. 2. Correlation of mitochondrial damage score with linearized passive urethral resistance relation (linPURR) grade.
FIG. 1. Transmission electron microscopy shows morphological changes in detrusor mitochondria according to severity of partial bladder outlet obstruction. A, most mitochondria in this grade 0 case had intact membrane, granules and no swelling. B to F, in grades 1 to 5 cases, respectively, mitochondria had progressive morphological changes, including loss of granules, swelling, disruption of inner membrane, condensation and clarification of matrix, and even disruption of outer membrane (arrowheads). Reduced from 327,720.
chondria per unit of cytoplasm area demonstrated no significant difference among the 3 groups (p 5 0.287, 0.367 and 0.338, respectively). DISCUSSION
In this study we observed that the mitochondrial damage score strongly correlated with the severity of partial bladder outlet obstruction (fig. 2). In our previous study we demonstrated that partial bladder outlet obstruction significantly decreased regional blood perfusion in the bladder.14 These results were consistent with the findings of Brading.15 We also noted a similar effect on energy metabolism after ischemia and bladder outlet obstruction.16, 17 Thus, it is conceivable that there are ischemic changes in the detrusor after bladder outlet obstruction develops. It is well known that anoxia and ischemia are important causes of cell injury and death. The detrusor may sustain ischemic injury and be subject to oxidative stress with subsequent mitochondrial damage and impaired bladder function. It is conceivable that in partial bladder outlet obstruction detrusor mitochondria are exposed to ischemia elicited reactive oxygen species and free radicals. In ischemia muscle mitochondria are vulnerable to oxidative damage due to incomplete oxygen reduction.18 Flameng et al reported that the
mitochondrial damage score increased significantly with severe ischemic injury of the human myocardium.10 They also suggested that disruption of the mitochondrial inner and outer membranes is a hallmark of irreversible ischemic injury. In 1997 Schirmer et al used the same mitochondrial damage score to describe morphological alterations in myocardial mitochondria after endocardial countershocks in dogs.11 They suggested that morphological changes may be associated with impaired mitochondrial function and oxidative metabolism. Our results also suggest structural destruction with possible functional impairment of the detrusor mitochondria as the severity of partial bladder outlet obstruction increases. These findings are consistent with the results of our previous studies,3, 19 in which we noted that the high energy phosphates, including phosphocreatine and adenosine triphosphate, were decreased in rat detrusor strips and the detrusor became fatigued as a result of energy consumption by frequent detrusor contractions.19 Furthermore, we reported that impaired mitochondrial respiratory function was accompanied by impaired detrusor contractile function in partial bladder outlet obstruction.3 Thus, mitochondrial structural damage may explain the possible impaired function and eventual fatigue of the detrusor in partial bladder outlet obstruction. Our results indicate that mean mitochondrial volume increased with the severity of partial bladder outlet obstruction (fig. 3). This finding was consistent with the mitochondrial damage score, which also reflected swelling in partial bladder outlet obstruction, and is in agreement with the morphological studies of Elbadawi et al.1, 2 However, mean mitochondrial volume was not different in the equivocal and obstructive groups. This finding suggests that there is a limit to the increase in mitochondrial size in severe partial bladder outlet obstruction, which is likely due to disruption of the mitochondrial outer membrane. It is well known that mitochondria are easily swollen artificially due to delay specimen fixation. Thus, the possibility of artificial mitochondrial swelling during specimen processing must be considered. One of us (S. H. L.) performed all biopsies and obtained the biopsy specimens of less than 1 mm.3, which were immediately fixed after removal of the tissue from the patients to prevent artificial mitochondrial swelling. Furthermore, most mitochondria in our linearized passive urethral resistance relation grade 0 cases were not swollen. Therefore, our measurement of mitochondrial size is reliable. On the other hand, there were no significant changes in mitochondrial volume density or surface density of the mito-
228
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TABLE 2. Mitochondrial damage score and mean mitochondrial volume after various degrees of partial bladder outlet obstruction, as defined by the International Continence Society nomogram No. Pts.
Mean 6 SE (95% CI)
No obstruction 14 0.516 6 0.132 (0.232–0.801) Equivocal result 14 1.31 6 0.080 (1.13–1.48) Obstruction 24 1.72 6 0.086 (1.55–1.90) p Value ,0.001 * Significant difference between all but these 2 groups using the Tukey method for post hoc multiple comparisons.
FIG. 3. Correlation of mean mitochondrial volume with linearized passive urethral resistance relation (linPURR) grade.
5.90 6 0.465 (4.890–6.900) 7.83 6 0.654 (6.420–9.244)* 8.13 6 0.492 (7.115–9.152)* 0.015
FIG. 5. Correlation of mitochondrial (mt) outer membrane surface density with linearized passive urethral resistance relation (linPURR) grade.
FIG. 4. Correlation of mitochondrial volume density with linearized passive urethral resistance relation (linPURR) grade.
chondrial outer membrane in partial bladder outlet obstruction. Barth et al stated that mitochondrial volume density may be an indicator of cell oxygen capacity.4 In addition, mitochondrial outer membrane surface density has been thought to correlate with the quantity of the enzyme system in the mitochondrial membrane and the compartment enclosed within it.6 Thus, our results suggest that there are no significant changes in the oxidative capacity and enzyme quantity of mitochondria in patients with various degrees of severity of partial bladder outlet obstruction. However, since mitochondria are easily swollen after injury, Bugge hypothesized that surface density of the mitochondrial inner membrane is a more valid indicator than mitochondrial volume density.5 Thus, the structural destruction changes of the mitochondrial inner membrane, as assessed by mitochondrial damage score, should be more meaningful for evaluat-
FIG. 6. Correlation of number of mitochondria per unit of cytoplasm area with linearized passive urethral resistance relation (linPURR) grade.
ing mitochondrial damage. However, our results showed that these 2 densities tended to increase as the severity of bladder outlet obstruction increased. This tendency may be a consequence of mitochondrial swelling rather than an increase of oxidative capacity or enzyme quantity. In this study the number of mitochondria per unit of cytoplasm area did not change significantly in partial bladder outlet obstruction. However, the tendency to decrease may also explain our previous findings of impaired mitochondrial function and detrusor contractile function in obstruction.3, 19 Furthermore, this tendency may result from mitochondrial degradation and explain detrusor fatigue in chronic partial bladder outlet obstruction. However, this possibility warrants further investigation.
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF DETRUSOR MITOCHONDRIA
According to our data only 3 cases were classified as grade 5 and none as grade 6. Theoretically patients with complete urinary retention should be classified as having severe bladder outlet obstruction. However, we excluded from study patients with urinary retention who had a urethral catheter since bladder outlet obstruction was completely relieved after catheterization. Therefore, only a few patients had severe bladder outlet obstruction in our study. The frequency distribution of the linearized passive urethral resistance relation in our study was similar to that of other reports.20, 21 Patient age distribution in the various obstructive groups was not significantly different except that those in the grade 0 obstructive group were younger than those in other groups. In previous studies we observed that mitochondrial DNA deletion and oxidative damage increased in various human tissues during aging.18 Our recent study revealed that aged rat detrusor muscle showed great fatigue and became fatigued easily by repeat detrusor contractions.19 Energy reserving and producing capability may decrease in the aged rat bladder.19 Thus, our results may be influenced by the factor of age. However, when subjecting our data to multiple regression analysis, age did not have a significant role in determining the changes of mitochondrial morphology in partial bladder outlet obstruction. Biopsy specimens obtained in this study should be representative, since they were obtained by performing 5 detrusor cold cup biopsies randomly from each lateral wall of the bladder in each case. Hailemariam et al claimed that the uniformity of bladder detrusor structural organization allows valid application of study criteria and protocols to small biopsies from various bladder detrusor sites.22 In regard to the appropriate sites of detrusor biopsies in our current study, the sampling should be appropriate due to the uniform organization of the detrusor. The inherent limitations of our study should be recognized. The data of duration of obstructive symptoms were obtained from a questionnaire, and answers were subjective and depended on patient memory. Thus, we focused on the severity of bladder outlet obstruction instead of duration as a correlate with morphological detrusor changes in partial bladder outlet obstruction. In addition, the urodynamic analysis was done at a single time point. Repeat pressure flow studies in an individual seemed to be inhumane due to invasiveness and unnecessary because of high reproducibility and accuracy.
4.
5.
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7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
CONCLUSIONS
Swelling and structural derangement of detrusor mitochondria are associated with the severity of partial bladder outlet obstruction. These changes may relate to impairment of respiratory function and oxidative metabolism in mitochondria during partial bladder outlet obstruction. Morphological and functional alterations of the detrusor mitochondria may explain voiding dysfunction.
17.
18. 19. 20.
Ling-Jen Tai assisted with the statistical analysis. REFERENCES
1. Elbadawi A., Yalla S. V. and Resnick N. M.: Structural basis of geriatric voiding dysfunction. IV. Bladder outlet obstruction. J Urol, 150: 1681, 1993. 2. Elbadawi, A.: Functional pathology of urinary bladder muscularis: the new frontier in diagnostic uropathology. Semin Diagn Pathol, 10: 314, 1993. 3. Lin, A. T.-L., Chen, K.-K., Yang, C.-H. et al: Effects of outlet
21.
22.
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obstruction and its reversal on mitochondrial enzyme activity in rabbit urinary bladders. J Urol, 160: 2258, 1998. Barth, E., Sta¨mmler, G., Speiser, B. et al: Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. Molec Cell Cardiol, 24: 669, 1992. Bugge, H. P.: Morphometry of mitochondria in ovine forestomach epithelia: high densities of mitochondria correlate with specialised metabolic activity. J Anat, 143: 65, 1985. Madhavi, C. and Jacob, M.: Morphometry of mitochondria in the choroidal ependyma of hydrocephalic guineapigs. Ind J Med Res, 96: 72, 1992. Elbadawi, A.: Ultrastructure of vesicourethral innervation I. Neuroeffector and cell junctions in the male internal sphincter. J Urol, 128: 180, 1982. Scha¨fer, W.: Analysis of bladder-outlet function with the linearized passive urethral resistance relation, linPURR, and a disease-specific approach for grading obstruction: from complex to simple. World J Urol, 13: 47, 1995. Griffiths, D., Hofner, K., van Mastrigt, R. et al: Standardization of terminology of lower urinary tract function: pressure-flow studies of voiding, urethral resistance, and urethral obstruction. International Continence Society Subcommittee on Standardization of Terminology of Pressure-Flow Studies. Neurourol Urodyn, 16: 1, 1997. Flameng, W., Borgers, M., Daenen, W. et al: Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J Thorac Cardiovasc Surg, 79: 413, 1980. Schirmer, U., Hemmer, W., Lindner, K. H. et al: Ultrastructural alterations in the right and left ventricular myocardium following multiple low energy endocardial countershocks in anesthetized dogs. PACE, 20: 79, 1997. Weibel, E. R., Kistler, G. S. and Scherle, W. F.: Practical stereological methods for morphometric cytology. J Cell Biol, 30: 23, 1966. Weibel, E. R. and Bolender, R. P.: Stereological techniques for electron microscopic morphometry. In.: Principles and Techniques of Electron Microscopy: Biological Applications. Edited by M. A. Hayat. New York: Van Nostrand Reinhold, vol. III, chapt. 6, pp. 237–296, 1973. Lin, A. T. L., Chen, M. T., Yang, C. H. et al: Blood flow of the urinary bladder— effects of outlet obstruction and correlation with energetic metabolism. Neurourol Urodyn, 14: 285, 1995. Brading, A. F.: Alterations in the physiological properties of urinary bladder smooth muscle caused by bladder emptying against an obstruction. Scand J Urol Nephrol, suppl, 184: 51, 1997. Lin, A. T.-L., Monson, F. C., Kato, K. et al: Effect of chronic ischemia on glucose metabolism of rabbit urinary bladder. J Urol, 142: 1127, 1989. Kato, K., Lin, A. T. L., Haugaard, N. et al: Effects of outlet obstruction on glucose metabolism of the rabbit urinary bladder. J Urol, 143: 844, 1990. Wei, Y. H.: Oxidative stress and mitochondrial DNA mutations in human aging. Proc Soc Exp Biol Med, 217: 53, 1998. Lin, A. T. L., Yang, C. H. and Chang, L. S.: Impact of aging on rat urinary bladder fatigue. J Urol, 157: 1990, 1997. Jensen, K. M., Jørgensen, J. B. and Mogensen, P.: Urodynamics in prostatism II. Prognostic value of pressure-flow study combined with stop-flow test. Scand J Urol Nephrol, suppl, 114: 72, 1988. Nitti, V. W., Kim, Y. and Combs, A. J.: Correlation of the AUA symptom index with urodynamics in patients with suspected benign prostatic hyperplasia. Neurourol Urodyn, 13: 521, 1994. Hailemariam, S., Elbadawi, A., Yalla, S. V. et al: Structural basis of geriatric voiding dysfunction. V. Standardized protocols for routine ultrastructural study and diagnosis of endoscopic detrusor biopsies. J Urol, 157: 1783, 1997.
Vol. 163, 225–229, January 2000 Printed in U.S.A.
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF HUMAN DETRUSOR MITOCHONDRIA WITH URODYNAMIC CORRELATION AFTER PARTIAL BLADDER OUTLET OBSTRUCTION SHING-HWA LU,* YAU-HUEI WEI, LUKE S. CHANG, ALEX T. L. LIN, KUANG-KUO CHEN AND AN-HANG YANG†,‡ From the Divisions of Urology (Department of Surgery) and Ultrastructural and Molecular Pathology (Department of Pathology), Veterans General Hospital-Taipei Institute of Clinical Medicine, School of Medicine and Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China
ABSTRACT
Purpose: We correlated ultrastructural changes in mitochondria in the human detrusor with the severity of partial bladder outlet obstruction on urodynamics. Materials and Methods: We recruited into the study 52 men with and without bladder outlet obstruction symptoms. The severity of partial bladder outlet obstruction was determined by pressure flow study. Random detrusor biopsy specimens obtained by cystoscopy were fixed immediately and processed for transmission electron microscopic observation. Random areas were photographed for further morphological and morphometric analysis using mitochondrial damage score and stereological principles. Results: Mitochondrial damage score and mean mitochondrial volume strongly correlated with the urodynamic severity of partial bladder outlet obstruction, while mitochondrial volume density, surface density of the mitochondrial outer membrane and number of mitochondria per unit of cytoplasm area did not significantly correlate with severity. Conclusions: Detrusor mitochondrial swelling and structural destruction increased with the severity of partial bladder outlet obstruction. These changes may be associated with impaired mitochondrial function and oxidative metabolism after partial bladder outlet obstruction. Detrusor mitochondrial damage may explain voiding dysfunction after partial bladder outlet obstruction develops. KEY WORDS: bladder, urodynamics, mitochondria, bladder neck obstruction
Bladder outlet obstruction causes significant morbidity, including irritative voiding symptoms, voiding difficulty and even urinary retention with or without detrusor failure. However, the pathogenesis of these disorders has not been fully elucidated. Some ultrastructural studies have revealed widely separated muscle cells with intermediate cell junction reduction, collagenosis and hypertrophic muscle cells in human and animal bladders after the development of bladder outlet obstruction.1, 2 Elbadawi et al also observed swollen, disrupted and vacuolated detrusor mitochondria in partial bladder outlet obstruction.1, 2 Recently we reported that impaired mitochondrial enzyme function has an important role in decreasing energy production and contractility of the detrusor in bladder outlet obstruction in rabbits.3 Furthermore, some groups have performed morphometry of the mitochondria in various tissues other than the detrusor in human and animals under different physiological or pathological conditions.4 – 6 However, to our knowledge there has been no quantitative study of human detrusor mitochondria in partial bladder outlet obstruction. We used stereological principles and mitochondrial damage score to evaluate morphological changes in detrusor mitochondria in various grades of partial bladder outlet obstruction severity, as determined by urodynamics.
MATERIALS AND METHODS
Patients and specimen processing. From December 1996 to November 1998, 67 men with and without obstructive voiding symptoms were evaluated in this study. Those without obstructive voiding symptoms had a ureteral stone, stricture or tumor. Partial obstruction was defined as bladder outlet obstruction that was not completely occlusive. Excluded from study were 15 patients with possbile neurogenic bladder, including those with stroke, spinal cord disease, diabetes mellitus, Parkinsonism and spinal bifida. A total of 52 men 34 to 79 years old (mean age 68.2) were enrolled in this study, and evaluated by the International Prostate Symptom Score (I-PSS) and quality of life assessment index (table 1). Written informed consent was obtained from each patient. Preoperatively 5 cold cup detrusor biopsies were performed randomly in each lateral bladder wall using spinal anesthesia. Each biopsy specimen was less than 1 mm.3. Specimens were immediately fixed with 2.5% glutaraldehyde in phosphate buffer, pH 7.4, for 2 hours at room temperature. They were thoroughly washed, post-fixed with 1% buffered osmium tetroxide, and subsequently processed with standard dehydration and embedding procedures. Ultrathin sections (70 nm.) were doubly stained with uranyl acetate and lead citrate,7 and examined with a transmission electron microscope at 60 kV. Urodynamic studies (table 1). Medication, such as antihistamine, that would interfere with detrusor function was discontinued for at least 3 weeks before urodynamics. In addition, patients with a urinary tract infection were excluded from study. Medium fill (30 ml. per minute) cystometry with a pressure flow study was performed at least 2 days before detrusor biopsy in each case. Cystometry was done by infus-
Accepted for publication August 13, 1999. * Recipient of Veterans General Hospital-Taipei and National Yang-Ming University Research Grants VGH-262, 1998 and VGHYM87-S1 to 08 1998. † Recipient of Veterans General Hospital-Taipei and National Yong-Ming University Grant VGH-360, 1998. ‡ Requests for reprints: Division of Ultrastructural and Molecular Pathology, Department of Pathology, Veterans General Hospital– Taipei, No. 201, Section 2, Shih Pai Rd., Taipei 112, Taiwan, Republic of China. 225
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TABLE 1. Clinical characteristics of patients with obstruction of various grades as defined by the linearized passive urethral resistance relation nomogram Mean 6 SD (range) 0 No. pts. Age Yrs. symptoms I-PSS (range 0–35) Quality of life assessment index (range 0–6) PSA (ng./ml.) Prostate size (ml.) Max. flow rate (ml./sec.) Vol. at initial urge (ml.) Bladder capacity (ml.) Post-void residual urine (ml.)
11 55.1 6 9.75 0.27 6 0.61 5.36 6 3.38 0.73 6 0.65
1 (34.0) (2.00) (12.0) (2.00)
11 72.7 6 4.31 3.86 6 2.81 14.5 6 5.50 3.64 6 1.03
(13.0) (9.00) (17.0) (3.00)
2
3
4
6 72.5 6 3.39 (9.00) 3.47 6 2.58 (6.80) 16.3 6 9.42 (23.0) 4.17 6 0.75 (2.00)
12 70.5 6 9.14 (33.0) 3.96 6 2.67 (9.00) 17.2 6 5.97 (17.0) 3.92 6 1.08 (3.00)
9 71.7 6 4.85 (14.0) 2.78 6 2.03 (5.00) 21.0 6 3.64 (13.0) 4.67 6 0.71 (2.00)
5 3 71.7 6 6.81 3.67 6 0.76 17.3 6 1.53 4.67 6 0.58
(13.0) (1.50) (3.00) (1.00)
1.21 6 0.74 (2.28) 3.45 6 2.60 (7.84) 4.07 6 2.18 (5.50) 3.48 6 1.26 (4.57) 3.34 6 1.90 (5.24) 4.91 6 2.59 (4.56) 24.1 6 3.75 (15.0) 36.1 6 20.5 (71.3) 39.3 6 15.1 (32.3) 37.2 6 22.2 (70.3) 57.3 6 25.9 (84.4) 41.1 6 18.7 (34.7) 20.1 6 4.81 (14.0) 9.00 6 2.57 (10.0) 7.00 6 2.76 (6.00) 8.75 6 2.56 (8.00) 7.89 6 2.32 (6.00) 9.00 6 2.65 (5.00) 253.1 6 75.2 (269) 152.5 6 96.3 (352) 200.0 6 87.9 (207) 156.7 6 47.3 (146) 123.8 6 67.9 (178) 164.3 6 144.7 (267) 397.6 6 125.9 (432) 197.0 6 107.2 (357) 264.5 6 89.3 (222) 231.3 6 69.7 (202) 184.6 6 86.4 (203) 244.7 6 186.1 (364) 8.18 6 7.51 (20.0) 21.0 6 12.2 (40.0) 16.7 6 12.5 (35.0) 53.3 6 79.4 (290) 100.0 6 73.0 (235) 116.7 6 76.4 (150)
ing sterile water at room temperature through an 8Fr urethral catheter. A 5Fr urethral catheter and rectal catheter were inserted and connected to fluid filled transducers at the level of the symphysis pubis to measure intravesical and abdominal pressure, respectively. Detrusor pressure was determined by subtracting abdominal from intravesical pressure. Bladder compliance was not significantly decreased. Subsequently we plotted the pressure flow study data on the linearized passive urethral resistance relation and International Continence Society nomograms8, 9 with patients classified into groups according to the severity of partial bladder outlet obstruction. All evaluations were performed by one of us (S. H. L.). For transmission electron microscopic observation 6 photographs of different fields were obtained randomly in each random biopsy specimen at 33,176 magnification. For each micrograph the whole area was reviewed and all mitochondria were evaluated. Magnification of all micrographs was calibrated using 2 carbon grating replicas. For mitochondrial score each negative at 33,176 magnification was examined under a dissecting microscopy at 3110 magnification by one of us (S. H. L.). Mitochondrial damage score was assessed blindly using the methods described by Flameng10 and Schirmer et al.11 Each mitochondrion was scored as grade 0 to 4 to represent the severity of structural destruction. Mean scores of all mitochondria (approximately 500) per case were then calculated. Morphometric analysis. Detrusor mitochondria were analyzed quantitatively by point counting.12, 13 Negatives were projected by an enlarger onto a screen covered with a sheet of square lattice of points at 3 mm. intervals and a sheet of test lines 3 mm. long with 2.6 mm. between 2 parallel test lines. Intersections of the square lattice lines or the end points of the test line were considered test points. Test points or test lines over the mitochondria, cytoplasm and outer mitochondrial surface were counted and totaled in each photograph. According to previously described stereological and morphometric principles, 6, 12, 13 certain formulas were used to estimate morphometric parameters. Mean mitochondrial volume, used to evaluate the size of individual mitochondria, was estimated by dividing the area or number of test points falling on the mitochondria (Pi) by the total number of mitochondria (Nt). Mitochondria volume density or volume fraction per unit volume of cytoplasm (Vv) was measured as Pi/Pc, where Pc represents the number of test points falling on the cytoplasm. Using the test line system surface density (Sv) of the mitochondrial outer membrane was estimated using the formula, Sv 5 4 3 Ii/Pc 3 LT, where Sv represents the mitochondrial outer membrane surface fraction per unit volume of cytoplasm, Ii the number of test line intersections with mitochondrial outer membrane and LT the test line length. The number of mitochondria per unit of cytoplasm area (NA) was calculated as Nt/Pc.
Statistics. We calculated the mean value plus or minus standard error and 95% confidence interval of the morphometric analysis and mitochondrial scores in each case. To evaluate the association of clinical data with transmission electron microscope findings we performed multiple linear regression and 1-way analysis of variance by the Tukey method for post hoc multiple comparisons using computer software with p ,0.05 considered statistically significant. RESULTS
According to the linearized passive urethral resistance relation nomogram 11 cases were categorized as grade 0, 11–grade 1, 6–grade 2, 12–grade 3, 9–grade 4 and 3–grade 5. Table 1 shows the urodynamics data. Transmission electron microscopy revealed progressive detrusor mitochondrial changes, including a loss of granules, swelling, condensation and clarification of the mitochondrial matrix, and disruption of the inner and outer membranes as the severity of partial bladder outlet obstruction increased (fig. 1). Mean mitochondrial score correlated well with the severity of bladder outlet obstruction, as determined by linearized passive urethral resistance relation obstructive grade8 (fig. 2). When patients were divided into nonobstructive, equivocal and obstructive groups according to the International Continence Society nomogram,9 mitochondrial score showed a statistically significant difference among the 3 groups (p ,0.001). Moreover, comparing any 2 groups revealed significant differences (p ,0.01, table 2). In addition, the mitochondrial score in each individual mitochondrion appeared different in the same cell of the detrusor. Mean mitochondrial score was higher in the 24 patients with than the 28 without detrusor instability (1.543 6 0.090 versus 1.066 6 0.136, p 5 0.007). Morphometric analysis revealed that the mean volume of mitochondria positively correlated with the severity of partial bladder outlet obstruction, as determined by linearized passive urethral resistance relation obstructive grade (fig. 3). Mitochondria volume density, surface density of the mitochondrial outer membrane and number of mitochondria per unit of cytoplasm area did not correlate significantly with the severity of partial bladder outlet obstruction, as classified by obstructive grade (figs. 4 to 6). When patients were divided into nonobstructive, equivocal and obstructive groups according to the International Continence Society nomogram, only mean mitochondrial volume revealed a statistically significant difference (p 5 0.015) among the 3 groups. Furthermore, when any 2 groups were compared, mean mitochondrial volume in the nonobstructive group was smaller than that in the equivocal or obstructive group (p ,0.05). However, the difference in the equivocal and obstructive groups was not statistically significant (p 5 0.917, table 2). Mitochondria volume density, surface density of the mitochondrial outer membrane and number of mito-
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF DETRUSOR MITOCHONDRIA
227
FIG. 2. Correlation of mitochondrial damage score with linearized passive urethral resistance relation (linPURR) grade.
FIG. 1. Transmission electron microscopy shows morphological changes in detrusor mitochondria according to severity of partial bladder outlet obstruction. A, most mitochondria in this grade 0 case had intact membrane, granules and no swelling. B to F, in grades 1 to 5 cases, respectively, mitochondria had progressive morphological changes, including loss of granules, swelling, disruption of inner membrane, condensation and clarification of matrix, and even disruption of outer membrane (arrowheads). Reduced from 327,720.
chondria per unit of cytoplasm area demonstrated no significant difference among the 3 groups (p 5 0.287, 0.367 and 0.338, respectively). DISCUSSION
In this study we observed that the mitochondrial damage score strongly correlated with the severity of partial bladder outlet obstruction (fig. 2). In our previous study we demonstrated that partial bladder outlet obstruction significantly decreased regional blood perfusion in the bladder.14 These results were consistent with the findings of Brading.15 We also noted a similar effect on energy metabolism after ischemia and bladder outlet obstruction.16, 17 Thus, it is conceivable that there are ischemic changes in the detrusor after bladder outlet obstruction develops. It is well known that anoxia and ischemia are important causes of cell injury and death. The detrusor may sustain ischemic injury and be subject to oxidative stress with subsequent mitochondrial damage and impaired bladder function. It is conceivable that in partial bladder outlet obstruction detrusor mitochondria are exposed to ischemia elicited reactive oxygen species and free radicals. In ischemia muscle mitochondria are vulnerable to oxidative damage due to incomplete oxygen reduction.18 Flameng et al reported that the
mitochondrial damage score increased significantly with severe ischemic injury of the human myocardium.10 They also suggested that disruption of the mitochondrial inner and outer membranes is a hallmark of irreversible ischemic injury. In 1997 Schirmer et al used the same mitochondrial damage score to describe morphological alterations in myocardial mitochondria after endocardial countershocks in dogs.11 They suggested that morphological changes may be associated with impaired mitochondrial function and oxidative metabolism. Our results also suggest structural destruction with possible functional impairment of the detrusor mitochondria as the severity of partial bladder outlet obstruction increases. These findings are consistent with the results of our previous studies,3, 19 in which we noted that the high energy phosphates, including phosphocreatine and adenosine triphosphate, were decreased in rat detrusor strips and the detrusor became fatigued as a result of energy consumption by frequent detrusor contractions.19 Furthermore, we reported that impaired mitochondrial respiratory function was accompanied by impaired detrusor contractile function in partial bladder outlet obstruction.3 Thus, mitochondrial structural damage may explain the possible impaired function and eventual fatigue of the detrusor in partial bladder outlet obstruction. Our results indicate that mean mitochondrial volume increased with the severity of partial bladder outlet obstruction (fig. 3). This finding was consistent with the mitochondrial damage score, which also reflected swelling in partial bladder outlet obstruction, and is in agreement with the morphological studies of Elbadawi et al.1, 2 However, mean mitochondrial volume was not different in the equivocal and obstructive groups. This finding suggests that there is a limit to the increase in mitochondrial size in severe partial bladder outlet obstruction, which is likely due to disruption of the mitochondrial outer membrane. It is well known that mitochondria are easily swollen artificially due to delay specimen fixation. Thus, the possibility of artificial mitochondrial swelling during specimen processing must be considered. One of us (S. H. L.) performed all biopsies and obtained the biopsy specimens of less than 1 mm.3, which were immediately fixed after removal of the tissue from the patients to prevent artificial mitochondrial swelling. Furthermore, most mitochondria in our linearized passive urethral resistance relation grade 0 cases were not swollen. Therefore, our measurement of mitochondrial size is reliable. On the other hand, there were no significant changes in mitochondrial volume density or surface density of the mito-
228
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF DETRUSOR MITOCHONDRIA
TABLE 2. Mitochondrial damage score and mean mitochondrial volume after various degrees of partial bladder outlet obstruction, as defined by the International Continence Society nomogram No. Pts.
Mean 6 SE (95% CI)
No obstruction 14 0.516 6 0.132 (0.232–0.801) Equivocal result 14 1.31 6 0.080 (1.13–1.48) Obstruction 24 1.72 6 0.086 (1.55–1.90) p Value ,0.001 * Significant difference between all but these 2 groups using the Tukey method for post hoc multiple comparisons.
FIG. 3. Correlation of mean mitochondrial volume with linearized passive urethral resistance relation (linPURR) grade.
5.90 6 0.465 (4.890–6.900) 7.83 6 0.654 (6.420–9.244)* 8.13 6 0.492 (7.115–9.152)* 0.015
FIG. 5. Correlation of mitochondrial (mt) outer membrane surface density with linearized passive urethral resistance relation (linPURR) grade.
FIG. 4. Correlation of mitochondrial volume density with linearized passive urethral resistance relation (linPURR) grade.
chondrial outer membrane in partial bladder outlet obstruction. Barth et al stated that mitochondrial volume density may be an indicator of cell oxygen capacity.4 In addition, mitochondrial outer membrane surface density has been thought to correlate with the quantity of the enzyme system in the mitochondrial membrane and the compartment enclosed within it.6 Thus, our results suggest that there are no significant changes in the oxidative capacity and enzyme quantity of mitochondria in patients with various degrees of severity of partial bladder outlet obstruction. However, since mitochondria are easily swollen after injury, Bugge hypothesized that surface density of the mitochondrial inner membrane is a more valid indicator than mitochondrial volume density.5 Thus, the structural destruction changes of the mitochondrial inner membrane, as assessed by mitochondrial damage score, should be more meaningful for evaluat-
FIG. 6. Correlation of number of mitochondria per unit of cytoplasm area with linearized passive urethral resistance relation (linPURR) grade.
ing mitochondrial damage. However, our results showed that these 2 densities tended to increase as the severity of bladder outlet obstruction increased. This tendency may be a consequence of mitochondrial swelling rather than an increase of oxidative capacity or enzyme quantity. In this study the number of mitochondria per unit of cytoplasm area did not change significantly in partial bladder outlet obstruction. However, the tendency to decrease may also explain our previous findings of impaired mitochondrial function and detrusor contractile function in obstruction.3, 19 Furthermore, this tendency may result from mitochondrial degradation and explain detrusor fatigue in chronic partial bladder outlet obstruction. However, this possibility warrants further investigation.
MORPHOLOGICAL AND MORPHOMETRIC ANALYSIS OF DETRUSOR MITOCHONDRIA
According to our data only 3 cases were classified as grade 5 and none as grade 6. Theoretically patients with complete urinary retention should be classified as having severe bladder outlet obstruction. However, we excluded from study patients with urinary retention who had a urethral catheter since bladder outlet obstruction was completely relieved after catheterization. Therefore, only a few patients had severe bladder outlet obstruction in our study. The frequency distribution of the linearized passive urethral resistance relation in our study was similar to that of other reports.20, 21 Patient age distribution in the various obstructive groups was not significantly different except that those in the grade 0 obstructive group were younger than those in other groups. In previous studies we observed that mitochondrial DNA deletion and oxidative damage increased in various human tissues during aging.18 Our recent study revealed that aged rat detrusor muscle showed great fatigue and became fatigued easily by repeat detrusor contractions.19 Energy reserving and producing capability may decrease in the aged rat bladder.19 Thus, our results may be influenced by the factor of age. However, when subjecting our data to multiple regression analysis, age did not have a significant role in determining the changes of mitochondrial morphology in partial bladder outlet obstruction. Biopsy specimens obtained in this study should be representative, since they were obtained by performing 5 detrusor cold cup biopsies randomly from each lateral wall of the bladder in each case. Hailemariam et al claimed that the uniformity of bladder detrusor structural organization allows valid application of study criteria and protocols to small biopsies from various bladder detrusor sites.22 In regard to the appropriate sites of detrusor biopsies in our current study, the sampling should be appropriate due to the uniform organization of the detrusor. The inherent limitations of our study should be recognized. The data of duration of obstructive symptoms were obtained from a questionnaire, and answers were subjective and depended on patient memory. Thus, we focused on the severity of bladder outlet obstruction instead of duration as a correlate with morphological detrusor changes in partial bladder outlet obstruction. In addition, the urodynamic analysis was done at a single time point. Repeat pressure flow studies in an individual seemed to be inhumane due to invasiveness and unnecessary because of high reproducibility and accuracy.
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CONCLUSIONS
Swelling and structural derangement of detrusor mitochondria are associated with the severity of partial bladder outlet obstruction. These changes may relate to impairment of respiratory function and oxidative metabolism in mitochondria during partial bladder outlet obstruction. Morphological and functional alterations of the detrusor mitochondria may explain voiding dysfunction.
17.
18. 19. 20.
Ling-Jen Tai assisted with the statistical analysis. REFERENCES
1. Elbadawi A., Yalla S. V. and Resnick N. M.: Structural basis of geriatric voiding dysfunction. IV. Bladder outlet obstruction. J Urol, 150: 1681, 1993. 2. Elbadawi, A.: Functional pathology of urinary bladder muscularis: the new frontier in diagnostic uropathology. Semin Diagn Pathol, 10: 314, 1993. 3. Lin, A. T.-L., Chen, K.-K., Yang, C.-H. et al: Effects of outlet
21.
22.
229
obstruction and its reversal on mitochondrial enzyme activity in rabbit urinary bladders. J Urol, 160: 2258, 1998. Barth, E., Sta¨mmler, G., Speiser, B. et al: Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. Molec Cell Cardiol, 24: 669, 1992. Bugge, H. P.: Morphometry of mitochondria in ovine forestomach epithelia: high densities of mitochondria correlate with specialised metabolic activity. J Anat, 143: 65, 1985. Madhavi, C. and Jacob, M.: Morphometry of mitochondria in the choroidal ependyma of hydrocephalic guineapigs. Ind J Med Res, 96: 72, 1992. Elbadawi, A.: Ultrastructure of vesicourethral innervation I. Neuroeffector and cell junctions in the male internal sphincter. J Urol, 128: 180, 1982. Scha¨fer, W.: Analysis of bladder-outlet function with the linearized passive urethral resistance relation, linPURR, and a disease-specific approach for grading obstruction: from complex to simple. World J Urol, 13: 47, 1995. Griffiths, D., Hofner, K., van Mastrigt, R. et al: Standardization of terminology of lower urinary tract function: pressure-flow studies of voiding, urethral resistance, and urethral obstruction. International Continence Society Subcommittee on Standardization of Terminology of Pressure-Flow Studies. Neurourol Urodyn, 16: 1, 1997. Flameng, W., Borgers, M., Daenen, W. et al: Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J Thorac Cardiovasc Surg, 79: 413, 1980. Schirmer, U., Hemmer, W., Lindner, K. H. et al: Ultrastructural alterations in the right and left ventricular myocardium following multiple low energy endocardial countershocks in anesthetized dogs. PACE, 20: 79, 1997. Weibel, E. R., Kistler, G. S. and Scherle, W. F.: Practical stereological methods for morphometric cytology. J Cell Biol, 30: 23, 1966. Weibel, E. R. and Bolender, R. P.: Stereological techniques for electron microscopic morphometry. In.: Principles and Techniques of Electron Microscopy: Biological Applications. Edited by M. A. Hayat. New York: Van Nostrand Reinhold, vol. III, chapt. 6, pp. 237–296, 1973. Lin, A. T. L., Chen, M. T., Yang, C. H. et al: Blood flow of the urinary bladder— effects of outlet obstruction and correlation with energetic metabolism. Neurourol Urodyn, 14: 285, 1995. Brading, A. F.: Alterations in the physiological properties of urinary bladder smooth muscle caused by bladder emptying against an obstruction. Scand J Urol Nephrol, suppl, 184: 51, 1997. Lin, A. T.-L., Monson, F. C., Kato, K. et al: Effect of chronic ischemia on glucose metabolism of rabbit urinary bladder. J Urol, 142: 1127, 1989. Kato, K., Lin, A. T. L., Haugaard, N. et al: Effects of outlet obstruction on glucose metabolism of the rabbit urinary bladder. J Urol, 143: 844, 1990. Wei, Y. H.: Oxidative stress and mitochondrial DNA mutations in human aging. Proc Soc Exp Biol Med, 217: 53, 1998. Lin, A. T. L., Yang, C. H. and Chang, L. S.: Impact of aging on rat urinary bladder fatigue. J Urol, 157: 1990, 1997. Jensen, K. M., Jørgensen, J. B. and Mogensen, P.: Urodynamics in prostatism II. Prognostic value of pressure-flow study combined with stop-flow test. Scand J Urol Nephrol, suppl, 114: 72, 1988. Nitti, V. W., Kim, Y. and Combs, A. J.: Correlation of the AUA symptom index with urodynamics in patients with suspected benign prostatic hyperplasia. Neurourol Urodyn, 13: 521, 1994. Hailemariam, S., Elbadawi, A., Yalla, S. V. et al: Structural basis of geriatric voiding dysfunction. V. Standardized protocols for routine ultrastructural study and diagnosis of endoscopic detrusor biopsies. J Urol, 157: 1783, 1997.