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Decrease in the Autonomic Innervation of Human Detrusor Muscle in Outflow Obstruction
0022-534 7/86/1362-0501$02.00/0
Vol. 136, August Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright © 1986 by The Williams & Wilkins Co.
DECREASE IN THE AUTONOMIC INNERVATION OF HUMAN DETRUSOR MUSCLE IN OUTFLOW OBSTRUCTION J. A. GOSLING,* S. A. GILPIN, J. S. DIXON AND C. J. GILPIN From the Department of Anatomy, University of Manchester, Manchester, United Kingdom
ABSTRACT
In a group of patients in whom bladder outflow obstruction had been confirmed urodynamically, quantitative assessment of the amount of autonomic nerve in detrusor biopsy samples has been carried out using light and electron microscope techniques. In each specimen allowance was made for muscle cell hypertrophy and increases in connective tissue, both of which occurred in response to bladder outflow obstruction. Similar quantitative assessment was performed on bladder biopsy samples from a group of unobstructed 'control' patients. When the results from the two groups were compared a statistically significant reduction in the amount of autonomic nerve supplying detrusor muscle was demonstrated in the obstructed group. This finding provides additional evidence that functional impairment of the urinary bladder occurs in response to outflow obstruction and emphasizes the need for prompt relief of the condition. Previous studies have demonstrated marked histological changes in the wall of the urinary bladder in response to outflow obstruction. 1- 6 These include infiltration of muscle bundles by connective tissue and smooth muscle cell hypertrophy. Additional studies using experimental animals have also indicated that the presence of outflow obstruction affects the nervous control of bladder activity. 7 However, the response of the innervation of the human detrusor to outflow obstruction has not been previously reported. Consequently, the present study has employed quantitative light and electron microscopic techniques to assess the amount of autonomic nerve in biopsy samples of bladder muscle obtained from a group of patients with urodynamically proven outflow obstruction. These results have been compared with those from a group of age-matched 'control' patients, who showed no urodynamic evidence of obstruction. MATERIALS AND METHODS
Patients. All patients included in the present study (23 males, 13 females) received detailed clinical, urodynamic and cystoscopic evaluation. Each patient had a stable cystometrogram and no evidence of neurological disease. For the purposes of ' the present study the patients were divided into two groups. The first group consisted of 19 patients (17 male, two female, , ranging in age from 58 to 76 years with a mean age of 69.5 years) in whom there was unequivocal urodynamic evidence of outflow obstruction (a detrusor pressure rise greater than 100 cm. of water in association with a flow rate of less than 10 ml./ sec.) accompanied by severe bladder trabeculation at cystoscopy. The second group consisted of 17 'control' patients (six males, 11 females, ranging in age from 58 to 79 years with a mean age of 65.9 years), in whom urodynamic assessment was normal and bladder trabeculation was judged cystoscopically to be absent. Patients in this group were being investigated for stress incontinence, interstitial cystitis (but with normal cystoscopy), bacterial cystitis or urothelial tumours. Tissue preparation for light microscopy. During endoscopy bladder biopsy samples from each patient were removed from the dome and/or lateral walls and rapidly frozen in 2-methylbutane cooled in liquid nitrogen. 15 µm. thick cryostat sections were prepared under standard conditions and adjacent sections
1
1
Accepted for publication February 27, 1986. * Requests for reprints: Department of Anatomy, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT, United Kingdom.
stained either for routine histology using Masson's trichrome stain or to demonstrate acetylcholinesterase-containing nerves using a modified Gomori method. 8 Tissue preparation for electron microscopy. Biopsy samples for electron microscopy were obtained from seven patients in the obstructed group (age range 67 to 74 years, mean age 71.0 years) and six patients in the control group (age range 60 to 72 years, mean age 66.8 years). Each biopsy sample was cut into one mm. 3 pieces and fixed in 2.5 per cent glutaraldehyde in sodium cacodylate buffer at 4C for two to 10 hours. After washing in cacodylate buffer alone the tissue pieces were postfixed in buffered osmium tetroxide. Following a further wash in buffer alone the tissue samples were stained en bloc with two per cent aqueous uranyl acetate before dehydration in ascending concentrations of acetone and embedding in epoxy resin. Thin sections were double stained with uranyl acetate and lead citrate prior to examination in a Philip's EM 300 electron microscope. Quantitative methods for light microscopy. (a) Amount of muscle per mm. 2 of tissue. The amount of muscle contained within a given area of detrusor tissue from each biopsy was determined by a point counting technique. Using a microscope eyepiece lattice graticule, the number of points which overlay muscle and those which overlay connective tissue were counted in multiple non-overlapping fields selected so as to cover the entire area of detrusor muscle present in each section. A mean value for the amount of muscle per mm. 2 of tissue was obtained for each biopsy and expressed as a percentage. (b) Assessment of muscle cell mean profile area (MPA). Photomicrographs of transversely sectioned smooth muscle cells were prepared and the total area of smooth muscle determined using a point counting technique. This area was then divided by the actual number of cells present to determine muscle cell MPA for each patient. (c) Amount of nerve per mm. 2 of tissue. Sections stained to demonstrate acetylcholinesterase-containing nerves were examined using the same magnification and the same eyepiece graticule as above. Counts were made of the number of points (grid intersections) which overlay enzyme-positive nerves in multiple non-overlapping fields selected so as to cover the entire area of muscle tissue contained in sections from each biopsy. Quantitative methods for electron microscopy. For each biopsy sample ten grid squares containing transversely-sectioned smooth muscle cells were selected and scanned at a magnification of X24,000. Counts were then made of the total number
502
GOSLING AND ASSOCIATES
of 'nerve profiles' present in these grid squares. When using the electron microscope a 'nerve profile' was defined as either i) two or more closely related axons and their associated neurilemmal cells, or ii) a single axon, with or without a covering of neurilemmal cell. After scanning each grid square, a micrograph was recorded at low magnification (X470) and used to determine the number of smooth muscle profiles. Using a point counting technique the total area of smooth muscle cell profiles was calculated and from this data a mean value for smooth muscle cell profile area was obtained. Statistical analysis of data. The data obtained from both the obstructed and control groups was found to be normally distributed using Filliben's test. Thus all statistical comparisons of data were performed using a two-tailed Student's t test. RESULTS
Amount of muscle per mm. 2 of tissue. The amount of smooth muscle per mm. 2 of tissue in the biopsies from the 19 patients with outflow obstruction ranged from 44 per cent to 71 per cent with a mean of 60 per cent± 2 per cent (S.E.) (table 1). The amount of smooth muscle per mm. 2 of tissue for each of the biopsies obtained from the 17 'control' patients ranged from 65 to 75 per cent with a mean of 69 ± 1 per cent (S.E.) (table 2).
When these two groups were compared, there was a significant decrease (p <0.01) in the amount of muscle per mm. 2 of tissue in the obstructed patients, due to the infiltration of the detrusor muscle bundles by connective tissue. For each biopsy the figure for the percentage of smooth muscle within a given area was used to convert the value for the amount of nerve per mm. 2 of tissue to the amount of nerve per mm. 2 of muscle. Nerve fibers per mm. 2 of detrusor muscle. Smooth muscle cell MPA for the obstructed group was 99 ± 8 µm. 2 compared with 48 ± 3 µm. 2 for the control group. Using electron microscopy a value of 45 ± 6 µm. 2 was obtained for the latter group. Since the two methods of measurement gave comparable results it was apparent that even cells with very small cross-sectional areas were not being overlooked at the light microscope level and thus this method of assessment was giving reliable data. Values of smooth muscle cell MP A from individual obstructed patients had increased by amounts varying from 19 per cent to 253 per cent when compared with the control group. Thus, for each biopsy from the obstructed group, an appropriate correction factor (the percentage increase in size) was applied when calculating the amount of nerve per mm. 2 of muscle. These results for each patient are expressed in tables 1 and 2. TABLE 1.
Patient No. 1 2 3 4 5 6 7 8 9
10
11 12 13 14 15 16 17 18 19
Group 1 (obstructed patients)
Age
Sex
Muscle/mm. 2 of Tissue
'Corrected' Nerves/mm. 2 of Muscle*
58 61 62 64 66 67 68 68 70 70 72 72 73 73 74 75 75 76 76
M M M M M F M M M M M M M F M M M M M
63% 61% 67% 60% 56% 68% 63% 51% 69% 62% 64% 44% 57% 58% 58% 44% 65% 52% 71%
131 33 33 38 160 127 227 367 257 133 350 34 340 247 194 7 46 46 65
* Corrected nerves/mm.' of muscle includes adjustment for cell hypertrophy.
TABLE 2.
Patient No. 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17
Group 2 ('control' patients)
Age
Sex
Muscle/mm. 2 of Tissue
Nerves/mm. 2 of Muscle
58 59 59 60 60 60 61 63 65 67 68 70 71 72 74 75 79
F M F F F M F F M F F M F M F F M
66% 70% 65% 66% 75% 71% 69% 72% 67% 68% 70% 66% 71% 69% 74% 71% 71%
331 406 418 250 433 455 343 466 192 396 255 308 258 475 314 440 236
The mean values of 'corrected' nerve per mm. 2 of muscle for the obstructed group showed a marked decrease [154 ± 27 (S.E.)] when compared to the control group [351 ± 22 (S.E.)] (see bar chart in fig. 1). The two sets of values were normally distributed and, when compared statistically (using Student's t test) the two groups were found to be significantly different (p <0.01). Electron microscopy. Results from obstructed patients showed a similar increase in smooth muscle cell mean profile area to those obtained using the light microscope. Consequently nerve profile counts for each biopsy from obstructed patients were adjusted by an appropriate factor to compensate for the increase in mean profile area. The results were expressed as the number of nerve profiles per mm. 2 of smooth muscle tissue and the data for seven obstructed patients and six control patients are shown in tables 3 and 4 respectively. Group mean values are illustrated in figure 2. The mean value obtained for the control group was 673 (± 72.6 S.E.) nerve profiles per mm. 2 of smooth muscle tissue. The obstructed group had a mean of 273 (± 90.2 S.E.) nerve profiles per mm. 2 of smooth muscle tissue, a value significantly lower than controls when using a two-tailed Student's t test (p <0.01). DISCUSSION
Previous studies have shown that bladder outflow obstruction is associated with hypertrophy of detrusor smooth muscle cells and infiltration of the muscularis by connective tissue. 1- 5 In the present study both these changes were observed in the biopsy samples from the obstructed patients when compared with controls. Thus in the quantitation of autonomic nerves supplying detrusor muscle tissue in the obstructed group, allowances have been made for the increased mean profile area of the smooth muscle cells and the increased amounts of connective tissue. These increases, if uncorrected, would lower the amount of nerve observed within a given unit area of tissue and give a spurious impression of a decrease in innervation. Consequently appropriate correction factors were applied to each biopsy from the obstructed group and when compared with the results from control patients a 56 per cent reduction in the amount of enzyme-positive nerves was demonstrated in the biopsies from obstructed bladders. The majority of the obstructed patients (17 out of 19) were male compared with only six out of 17 from the control group. However it has recently been shown that there is no significant difference in the amount of nerve per mm. 2 of detrusor muscle between age-matched normal males and females 9 and so the results of the present study cannot merely be explained as a sex difference in autonomic innervation. The light microscope method used in this study relies on a histochemical method to demonstrate acetylcholinesterase activity associated with autonomic nerve fibers and it could be argued that the observed reduction in the amount of nerve in
NERVE REDUCTION WITH OUTFLOW OBSTRUCTION
400
300
200
100
Obstructed
Control
Group
Group
FIG. 1. Effect of outflow obstruction on amount of nerve per mm. 2 of detrusor muscle measured by light microscopy; significantly different from controls (p <0.01). TABLE
Patient
Age
1 2 3 4 5 6 7
67 68 70 72 73 73 74
3. Obstructed patients Nerve Profiles/ mm. 2 Smooth Muscle*
Sex
191 396 155 14 537 596 19
F M M M M
F M
* Adjusted for increase in mean profile area. TABLE
4. Control patients
Patient
Age
Sex
1 2 3
60 61 67 70 71
F F F
4 5 6
72
Nerve Profiles/mm. 2 Smooth Muscle 492 451 924 705 782 685
M
F M
BOO
--
700
600
500
400
503
using portions of the same biopsy samples from selected patients in both groups. Counts of all types of autonomic nerve profiles per mm. 2 of smooth muscle (corrected for hypertrophy where applicable) were obtained for each biopsy and when the two groups were compared, a significant reduction (of about 60 per cent) in nerve density was demonstrated in the obstructed group. Thus the e.m. results endorse those of the light microscope study and confirm that the presence of outflow obstruction significantly reduces the amount of autonomic nerve supplying detrusor muscle. Most values for the amount of nerve in the obstructed patients (obtained by both light and e.m.) fall below the range obtained from control specimens. However the amount of nerve in a minority of specimens from the obstructed group falls within the control range. One possible explanation for these results is that the onset of the obstruction in such patients is relatively recent. On this basis the reduction in autonomic innervation would be less in these patients than in others in whom outflow obstruction was of longer duration. In this context it has recently been shown that the response of the detrusor to experimentally induced outlet obstruction is proportional to the degree of obstruction. 10 However an alternative explanation for the present results is that the detrusor muscle in some patients is richly innervated prior to the onset of the obstruction and, although the amounts of nerve have reduced, at the time of examination the levels remain within the control range. Future long-term studies on the same patients are required in order to examine this possibility. The significant reduction in innervation of the detrusor muscle in response to outflow obstruction may markedly impair the neuromuscular control of the bladder. In this regard it is interesting to note that various pharmacological changes compatible with this effect have been reported in experimentally induced bladder outflow obstruction. Furthermore recent unpublished studies by Brading and co-workers have demonstrated "denervation supersensitivity" in samples of detrusor muscle obtained from patients with outflow obstruction. It remains to be determined whether the changes in autonomic innervation presently demonstrated play a part in the etiology of bladder instability which frequently accompanies outflow obstruction. Reduction in the amount of nerve in the detrusor in response to outflow obstruction must be borne in mind when interpreting data from patients with accompanying neurological lesions. In such cases it will be difficult to distinguish primary neurological changes in the bladder wall from those which occur as a secondary response to the coexisting obstruction. Finally it is not known whether the reduction in the density of autonomic nerve supplying the bladder wall is altered following relief of the obstruction. Nevertheless the results of the present study clearly emphasise the need for early therapy to relieve the obstruction and thereby minimise adverse effects upon the autonomic innervation of the bladder.
300
REFERENCES 200
100
Obstructed
Control
Group
Croup
FIG. 2. Effect of outflow obstruction on number of nerve profiles per mm. 2 of detrusor muscle counted by electron microscopy; significantly different from controls (p <0.01).
bladders with outflow obstruction merely reflects decreased levels of enzyme activity and is not due to an actual reduction in autonomic innervation. In order to exclude this possibility parallel studies using the electron microscope were carried out
1. Brent, L. and Stephen, F. D.: The response of smooth muscle cells in the rabbit bladder to outflow obstruction. Invest. Urol., 12: 494, 1975. 2. Susset, J. G.: Effects of ageing and prostatic obstruction on detrusor morphology and function. In: Benign Prostatic Hypertrophy. Edited by Hinman, F. Springer-Verlag, New York, 1983. 3. Gilpin, S. A., Gosling, J. A. and Barnard, R. J.: Morphological and morphometric studies of the human obstructed, trabeculated urinary bladder. Br. J. Urol., 57: 525, 1986. 4. Levin, R. M., High, J. and Wein, A. J.: The effect of short-term obstruction on urinary bladder function in the rabbit. J. Urol., 132: 789, 1984. 5. Gosling, J. A. and Dixon, J. S.: Structure of trabeculated detrusor smooth muscle in cases of prostatic hypertrophy. Urol. Int., 35: 351, 1980. 6. Uvelius, B., Persson, L. and Mattiasson, A.: Smooth muscle cell hypertrophy and hyperplasia in the rat detrusor after short-time
504
GOSLING AND ASSOCIATES
infravesical outflow obstruction. J. Ural., 131: 183, 1984. 7. Uvelius, B., Ekstrom, J., Larsson, B. and Mattiasson, A.: Changes in the nervous control of the rat urinary bladder subjected to infravesical outflow obstruction. Proceedings of the International Continence Society, 14th Annual Meeting, p. 206, 1984. 8. Gomori, G.: Microscopic Histochemistry: Principles and Practice. University of Chicago Press, Chicago, 1952.
9. Gilpin, S. A., Gilpin, C. J., Dixon, J. S., Gosling, J. A. and Kirby, R. S.: The effect of age on the autonomic innervation of the urinary bladder. Br. J. Ural. (In press.) 10. Ghoniem, G. M., Regnier, C. H. and Susset, J. G.: The importance of the degree of outlet obstruction in detrusor response. Proceedings of the International Continence Society, 14th Annual Meeting, p. 211, 1984.
Vol. 136, August Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright © 1986 by The Williams & Wilkins Co.
DECREASE IN THE AUTONOMIC INNERVATION OF HUMAN DETRUSOR MUSCLE IN OUTFLOW OBSTRUCTION J. A. GOSLING,* S. A. GILPIN, J. S. DIXON AND C. J. GILPIN From the Department of Anatomy, University of Manchester, Manchester, United Kingdom
ABSTRACT
In a group of patients in whom bladder outflow obstruction had been confirmed urodynamically, quantitative assessment of the amount of autonomic nerve in detrusor biopsy samples has been carried out using light and electron microscope techniques. In each specimen allowance was made for muscle cell hypertrophy and increases in connective tissue, both of which occurred in response to bladder outflow obstruction. Similar quantitative assessment was performed on bladder biopsy samples from a group of unobstructed 'control' patients. When the results from the two groups were compared a statistically significant reduction in the amount of autonomic nerve supplying detrusor muscle was demonstrated in the obstructed group. This finding provides additional evidence that functional impairment of the urinary bladder occurs in response to outflow obstruction and emphasizes the need for prompt relief of the condition. Previous studies have demonstrated marked histological changes in the wall of the urinary bladder in response to outflow obstruction. 1- 6 These include infiltration of muscle bundles by connective tissue and smooth muscle cell hypertrophy. Additional studies using experimental animals have also indicated that the presence of outflow obstruction affects the nervous control of bladder activity. 7 However, the response of the innervation of the human detrusor to outflow obstruction has not been previously reported. Consequently, the present study has employed quantitative light and electron microscopic techniques to assess the amount of autonomic nerve in biopsy samples of bladder muscle obtained from a group of patients with urodynamically proven outflow obstruction. These results have been compared with those from a group of age-matched 'control' patients, who showed no urodynamic evidence of obstruction. MATERIALS AND METHODS
Patients. All patients included in the present study (23 males, 13 females) received detailed clinical, urodynamic and cystoscopic evaluation. Each patient had a stable cystometrogram and no evidence of neurological disease. For the purposes of ' the present study the patients were divided into two groups. The first group consisted of 19 patients (17 male, two female, , ranging in age from 58 to 76 years with a mean age of 69.5 years) in whom there was unequivocal urodynamic evidence of outflow obstruction (a detrusor pressure rise greater than 100 cm. of water in association with a flow rate of less than 10 ml./ sec.) accompanied by severe bladder trabeculation at cystoscopy. The second group consisted of 17 'control' patients (six males, 11 females, ranging in age from 58 to 79 years with a mean age of 65.9 years), in whom urodynamic assessment was normal and bladder trabeculation was judged cystoscopically to be absent. Patients in this group were being investigated for stress incontinence, interstitial cystitis (but with normal cystoscopy), bacterial cystitis or urothelial tumours. Tissue preparation for light microscopy. During endoscopy bladder biopsy samples from each patient were removed from the dome and/or lateral walls and rapidly frozen in 2-methylbutane cooled in liquid nitrogen. 15 µm. thick cryostat sections were prepared under standard conditions and adjacent sections
1
1
Accepted for publication February 27, 1986. * Requests for reprints: Department of Anatomy, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT, United Kingdom.
stained either for routine histology using Masson's trichrome stain or to demonstrate acetylcholinesterase-containing nerves using a modified Gomori method. 8 Tissue preparation for electron microscopy. Biopsy samples for electron microscopy were obtained from seven patients in the obstructed group (age range 67 to 74 years, mean age 71.0 years) and six patients in the control group (age range 60 to 72 years, mean age 66.8 years). Each biopsy sample was cut into one mm. 3 pieces and fixed in 2.5 per cent glutaraldehyde in sodium cacodylate buffer at 4C for two to 10 hours. After washing in cacodylate buffer alone the tissue pieces were postfixed in buffered osmium tetroxide. Following a further wash in buffer alone the tissue samples were stained en bloc with two per cent aqueous uranyl acetate before dehydration in ascending concentrations of acetone and embedding in epoxy resin. Thin sections were double stained with uranyl acetate and lead citrate prior to examination in a Philip's EM 300 electron microscope. Quantitative methods for light microscopy. (a) Amount of muscle per mm. 2 of tissue. The amount of muscle contained within a given area of detrusor tissue from each biopsy was determined by a point counting technique. Using a microscope eyepiece lattice graticule, the number of points which overlay muscle and those which overlay connective tissue were counted in multiple non-overlapping fields selected so as to cover the entire area of detrusor muscle present in each section. A mean value for the amount of muscle per mm. 2 of tissue was obtained for each biopsy and expressed as a percentage. (b) Assessment of muscle cell mean profile area (MPA). Photomicrographs of transversely sectioned smooth muscle cells were prepared and the total area of smooth muscle determined using a point counting technique. This area was then divided by the actual number of cells present to determine muscle cell MPA for each patient. (c) Amount of nerve per mm. 2 of tissue. Sections stained to demonstrate acetylcholinesterase-containing nerves were examined using the same magnification and the same eyepiece graticule as above. Counts were made of the number of points (grid intersections) which overlay enzyme-positive nerves in multiple non-overlapping fields selected so as to cover the entire area of muscle tissue contained in sections from each biopsy. Quantitative methods for electron microscopy. For each biopsy sample ten grid squares containing transversely-sectioned smooth muscle cells were selected and scanned at a magnification of X24,000. Counts were then made of the total number
502
GOSLING AND ASSOCIATES
of 'nerve profiles' present in these grid squares. When using the electron microscope a 'nerve profile' was defined as either i) two or more closely related axons and their associated neurilemmal cells, or ii) a single axon, with or without a covering of neurilemmal cell. After scanning each grid square, a micrograph was recorded at low magnification (X470) and used to determine the number of smooth muscle profiles. Using a point counting technique the total area of smooth muscle cell profiles was calculated and from this data a mean value for smooth muscle cell profile area was obtained. Statistical analysis of data. The data obtained from both the obstructed and control groups was found to be normally distributed using Filliben's test. Thus all statistical comparisons of data were performed using a two-tailed Student's t test. RESULTS
Amount of muscle per mm. 2 of tissue. The amount of smooth muscle per mm. 2 of tissue in the biopsies from the 19 patients with outflow obstruction ranged from 44 per cent to 71 per cent with a mean of 60 per cent± 2 per cent (S.E.) (table 1). The amount of smooth muscle per mm. 2 of tissue for each of the biopsies obtained from the 17 'control' patients ranged from 65 to 75 per cent with a mean of 69 ± 1 per cent (S.E.) (table 2).
When these two groups were compared, there was a significant decrease (p <0.01) in the amount of muscle per mm. 2 of tissue in the obstructed patients, due to the infiltration of the detrusor muscle bundles by connective tissue. For each biopsy the figure for the percentage of smooth muscle within a given area was used to convert the value for the amount of nerve per mm. 2 of tissue to the amount of nerve per mm. 2 of muscle. Nerve fibers per mm. 2 of detrusor muscle. Smooth muscle cell MPA for the obstructed group was 99 ± 8 µm. 2 compared with 48 ± 3 µm. 2 for the control group. Using electron microscopy a value of 45 ± 6 µm. 2 was obtained for the latter group. Since the two methods of measurement gave comparable results it was apparent that even cells with very small cross-sectional areas were not being overlooked at the light microscope level and thus this method of assessment was giving reliable data. Values of smooth muscle cell MP A from individual obstructed patients had increased by amounts varying from 19 per cent to 253 per cent when compared with the control group. Thus, for each biopsy from the obstructed group, an appropriate correction factor (the percentage increase in size) was applied when calculating the amount of nerve per mm. 2 of muscle. These results for each patient are expressed in tables 1 and 2. TABLE 1.
Patient No. 1 2 3 4 5 6 7 8 9
10
11 12 13 14 15 16 17 18 19
Group 1 (obstructed patients)
Age
Sex
Muscle/mm. 2 of Tissue
'Corrected' Nerves/mm. 2 of Muscle*
58 61 62 64 66 67 68 68 70 70 72 72 73 73 74 75 75 76 76
M M M M M F M M M M M M M F M M M M M
63% 61% 67% 60% 56% 68% 63% 51% 69% 62% 64% 44% 57% 58% 58% 44% 65% 52% 71%
131 33 33 38 160 127 227 367 257 133 350 34 340 247 194 7 46 46 65
* Corrected nerves/mm.' of muscle includes adjustment for cell hypertrophy.
TABLE 2.
Patient No. 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17
Group 2 ('control' patients)
Age
Sex
Muscle/mm. 2 of Tissue
Nerves/mm. 2 of Muscle
58 59 59 60 60 60 61 63 65 67 68 70 71 72 74 75 79
F M F F F M F F M F F M F M F F M
66% 70% 65% 66% 75% 71% 69% 72% 67% 68% 70% 66% 71% 69% 74% 71% 71%
331 406 418 250 433 455 343 466 192 396 255 308 258 475 314 440 236
The mean values of 'corrected' nerve per mm. 2 of muscle for the obstructed group showed a marked decrease [154 ± 27 (S.E.)] when compared to the control group [351 ± 22 (S.E.)] (see bar chart in fig. 1). The two sets of values were normally distributed and, when compared statistically (using Student's t test) the two groups were found to be significantly different (p <0.01). Electron microscopy. Results from obstructed patients showed a similar increase in smooth muscle cell mean profile area to those obtained using the light microscope. Consequently nerve profile counts for each biopsy from obstructed patients were adjusted by an appropriate factor to compensate for the increase in mean profile area. The results were expressed as the number of nerve profiles per mm. 2 of smooth muscle tissue and the data for seven obstructed patients and six control patients are shown in tables 3 and 4 respectively. Group mean values are illustrated in figure 2. The mean value obtained for the control group was 673 (± 72.6 S.E.) nerve profiles per mm. 2 of smooth muscle tissue. The obstructed group had a mean of 273 (± 90.2 S.E.) nerve profiles per mm. 2 of smooth muscle tissue, a value significantly lower than controls when using a two-tailed Student's t test (p <0.01). DISCUSSION
Previous studies have shown that bladder outflow obstruction is associated with hypertrophy of detrusor smooth muscle cells and infiltration of the muscularis by connective tissue. 1- 5 In the present study both these changes were observed in the biopsy samples from the obstructed patients when compared with controls. Thus in the quantitation of autonomic nerves supplying detrusor muscle tissue in the obstructed group, allowances have been made for the increased mean profile area of the smooth muscle cells and the increased amounts of connective tissue. These increases, if uncorrected, would lower the amount of nerve observed within a given unit area of tissue and give a spurious impression of a decrease in innervation. Consequently appropriate correction factors were applied to each biopsy from the obstructed group and when compared with the results from control patients a 56 per cent reduction in the amount of enzyme-positive nerves was demonstrated in the biopsies from obstructed bladders. The majority of the obstructed patients (17 out of 19) were male compared with only six out of 17 from the control group. However it has recently been shown that there is no significant difference in the amount of nerve per mm. 2 of detrusor muscle between age-matched normal males and females 9 and so the results of the present study cannot merely be explained as a sex difference in autonomic innervation. The light microscope method used in this study relies on a histochemical method to demonstrate acetylcholinesterase activity associated with autonomic nerve fibers and it could be argued that the observed reduction in the amount of nerve in
NERVE REDUCTION WITH OUTFLOW OBSTRUCTION
400
300
200
100
Obstructed
Control
Group
Group
FIG. 1. Effect of outflow obstruction on amount of nerve per mm. 2 of detrusor muscle measured by light microscopy; significantly different from controls (p <0.01). TABLE
Patient
Age
1 2 3 4 5 6 7
67 68 70 72 73 73 74
3. Obstructed patients Nerve Profiles/ mm. 2 Smooth Muscle*
Sex
191 396 155 14 537 596 19
F M M M M
F M
* Adjusted for increase in mean profile area. TABLE
4. Control patients
Patient
Age
Sex
1 2 3
60 61 67 70 71
F F F
4 5 6
72
Nerve Profiles/mm. 2 Smooth Muscle 492 451 924 705 782 685
M
F M
BOO
--
700
600
500
400
503
using portions of the same biopsy samples from selected patients in both groups. Counts of all types of autonomic nerve profiles per mm. 2 of smooth muscle (corrected for hypertrophy where applicable) were obtained for each biopsy and when the two groups were compared, a significant reduction (of about 60 per cent) in nerve density was demonstrated in the obstructed group. Thus the e.m. results endorse those of the light microscope study and confirm that the presence of outflow obstruction significantly reduces the amount of autonomic nerve supplying detrusor muscle. Most values for the amount of nerve in the obstructed patients (obtained by both light and e.m.) fall below the range obtained from control specimens. However the amount of nerve in a minority of specimens from the obstructed group falls within the control range. One possible explanation for these results is that the onset of the obstruction in such patients is relatively recent. On this basis the reduction in autonomic innervation would be less in these patients than in others in whom outflow obstruction was of longer duration. In this context it has recently been shown that the response of the detrusor to experimentally induced outlet obstruction is proportional to the degree of obstruction. 10 However an alternative explanation for the present results is that the detrusor muscle in some patients is richly innervated prior to the onset of the obstruction and, although the amounts of nerve have reduced, at the time of examination the levels remain within the control range. Future long-term studies on the same patients are required in order to examine this possibility. The significant reduction in innervation of the detrusor muscle in response to outflow obstruction may markedly impair the neuromuscular control of the bladder. In this regard it is interesting to note that various pharmacological changes compatible with this effect have been reported in experimentally induced bladder outflow obstruction. Furthermore recent unpublished studies by Brading and co-workers have demonstrated "denervation supersensitivity" in samples of detrusor muscle obtained from patients with outflow obstruction. It remains to be determined whether the changes in autonomic innervation presently demonstrated play a part in the etiology of bladder instability which frequently accompanies outflow obstruction. Reduction in the amount of nerve in the detrusor in response to outflow obstruction must be borne in mind when interpreting data from patients with accompanying neurological lesions. In such cases it will be difficult to distinguish primary neurological changes in the bladder wall from those which occur as a secondary response to the coexisting obstruction. Finally it is not known whether the reduction in the density of autonomic nerve supplying the bladder wall is altered following relief of the obstruction. Nevertheless the results of the present study clearly emphasise the need for early therapy to relieve the obstruction and thereby minimise adverse effects upon the autonomic innervation of the bladder.
300
REFERENCES 200
100
Obstructed
Control
Group
Croup
FIG. 2. Effect of outflow obstruction on number of nerve profiles per mm. 2 of detrusor muscle counted by electron microscopy; significantly different from controls (p <0.01).
bladders with outflow obstruction merely reflects decreased levels of enzyme activity and is not due to an actual reduction in autonomic innervation. In order to exclude this possibility parallel studies using the electron microscope were carried out
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