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Cavernous Oxygen Tension and Smooth Muscle Fibers: Relation and Function
~-6347/95/1545-1736$03.00/0
Vol. 154, 1736-1739. November 1995 Printed in U.S.A.
THE JOURNAL OF UR0UX;Y Copyright 0 1995 by AMERICAN U R O ~ I CASS~CLM'ION, AL INC.
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS: RELATION AND FUNCTION AHMED A. SA'ITAR, GEORGES SALPIGIDES, JEAN-JACQUES VANDERHAEGHEN, CLAUDE C. SCHULMAN AND ERIC WESPES" From the Department of Urology, Erasme Hospital and Department of Neuropeptides, Faculty of Medicine, University Clinics of Brussels, Brussels, Belgium
ABSTRACT
Purpose: We studied the effect of intracavernous oxygen tension on the alteration of cavernous smooth muscle fibers in potent and impotent men. Materials and Methods: Intracavernous oxygen tension (mm. Hg) was measured during flaccidity and 10 minutes after intracavernous prostaglandin E l injection in psychogenic control patients, and those with venous leakage and arterial lesions. Cavernous biopsies were performed and the percent of smooth muscle fibers was analyzed objectively using immunohistochemical actin anti-actin staining. Simultaneously brachial oxygen tension (mm. Hg) was measured and the cavernous brachial oxygen tension index was then determined. Results: At flaccidity no significant difference was noted in oxygen tension values among the 3 groups of patients. After prostaglandin E l injection cavernous oxygen tension and the cavernous brachial oxygen tension index in the control group were significantly different (p <0.01)from those of the venogenic and arteriogenic groups (p cO.01).The mean percent of cavernous smooth muscle fibers in the control group was sigdicantly different from those of the venous leakage and arterial lesion groups (p <0.01). There was a good correlation between the percent of cavernous muscle fibers and the value of oxygen tension before (p <0.05)and after prostaglandin E l injection (p CO.01). A similar correlation was noted between cavernous muscle fibers and cavernous brachial oxygen tension index in the different groups of patients (p <0.01). Conclusions: Reduction of the intracavernous smooth muscle fibers in impotent patients could be explained by low intracavernous oxygen tension. KEYWORDS:penis; impotence; blood gas monitoring, transcutaneous; prostaglandins E;muscle, smooth Penile erection results from increased arterial flow, sinusoidal smooth muscle relaxation and decreased venous return.1 Impaired relaxation of the cavernous smooth muscle fibers or alteration in its density represents the structural basis for erectile dysfunction.2 Previously it has been demonstrated in animal models that cavernous muscles can be altered by toxic or neurological factors.= Recently the effect of the ischemic factor or low oxygen on cavernous smooth muscles has been studied. It has been shown that low oxygen tension inhibits the release of the endothelium-derived relaxing factor (nitric oxide), the novel neurotransmitter for erection.6a7 Subsequently this low oxygen tension impaired the relaxation of the cavernous trabecular smooth muscle. However, the causes responsible for cavernous smooth muscle fiber alterations were not demonstrated. To study the effect of oxygen on the alteration of cavernous smooth muscle cells we measured the cavernous oxygen level (oxygen tension in mm. Hg) before and 10 minutes after intracavernous injection of 20 pg. prostaglandin E l in psychogenically impotent patients as a control group and impotent patients presenting with a venous leak or arterial lesion. Results were compared with intracavernous muscular quantity. Cavernous biopsies were analyzed objectively and the percent of smooth muscle fibers was determined.
64 years, mean 47) with venogenic impotence and 10 (age 38
to 63 years, mean 52) with arteriogenic impotence. Clinical evaluation. Patients underwent a complete impotence evaluation, including a detailed sequelae history and
physical examination. They were hospitalized for at least 1 night to evaluate nocturnal penile tumescence using the Rigiscant device. The RigiScan did not show normal nocturnal penile tumescence in the impotent patients. Those with pure venous leakage had a flow rate necessary to maintain an artificial erection of greater than 15 ml. per minute after intracavernous injection of 20 pg. prostaglandin Full blood and hormonal assays were done to exclude endocrinological disease. The bulbocavernous reflex was evaluated to exclude a neurological disorder. The penile brachial index was normal and penile pharmacoangiography did not reveal arterial insuficiency.9 Patients diagnosed as having pure arterial disease had abnormal brachial penile indexes. Pharmacoangiography confirmed arterial lesions. Pharmacocavernosometry-pharmacocavernosography was considered to be normal. Based on these evaluations disease was diagnosed as psychogenic in 5 patients, venogenic in 9 and arteriogenic in 10. Analysis of cavernous and brachial blood gases. Intracavernous blood was obtained by aspiration through a 19 gauge needle and a 2 ml. syringe. Blood gases were immediately MATERIALS A N D METHODS measured using an instrumentation laboratory (pH blood gas A total of 24 patients were enrolled in this study and analyzer). Simultaneously brachial (arterial) blood was obclassified into 3 groups, including 5 patients (age 29 to 41 tained and blood gases were immediately measured as deyears, mean 35.4)with psychogenic impotence, 9 (age 34 to scribed. Injection of 20 pg. prostaglandin E l was done using a 27% gauge needle inserted in the contralateral cavernous body. Ten minutes after injection cavernous blood was obAccepted for publication April 7, 1995. * Requests for re rints: Department of Urology, Erasme Hospital, 808 route de Lenni!, 1070 Brussels, Belgium. .t Dacomed. Minneapolis, Minnesota. 1736
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS
tained as described and blood gases were immediately measwed using the same analyzer. S p c i m e n and tissue processing. All patients underwent avernous biopsy. Specimens were immediately fmed in 10% formalin phosphate buffer solution before parafin embedding. The fixed tissue sections were processed through xylene embedded in paraffin and sectioned at 5 pm. One tissue section was stained with hematoxylin and eosin, and examined by light microscopy to evaluate the cavernous tissue pathologkally. Another tissue section was stained with actin anti-actin for immunohistochemical studies to determine the presence of smooth muscle cells.10 Immunohistochemical staining was done according to the biotin-streptavidin-peroxidase technique. Sections were incubated for 15minutes with 2% (volume-in-volume) hydrogen peroxide solution to block endogenous peroxidase activity and processed for immunohistochemical study. After incubation with 1:20 normal sheep serum the sections were incubated overnight (14 to 18 hours) with mouse anti-human anti-actin antibodies in a dilution of 150. The sections were rinsed and incubated for 30 minutes at room temperature successively with sheep anti-mouse immunoglobulin biotin diluted to 1500 and streptavidin, which was coupled to peroxidase (1:1,000) for 30 minutes. Peroxidase activity was revealed by 10 mg./lO ml. 3.3-diaminobenzidine, pH 7.4, containing 0.01% (volumein-volume) hydrogen peroxide for 5 to 10 minutes at room temperature. Computerized image analysis. Quantitative analysis of smooth muscle fibers in cavernous tissue was determined using an image analyzer system combined with a light microscope equipped with a video camera. Images were interactively discriminated and measurements were obtained from the resulting binary images. The percent of smooth muscle cells for each image results from the quantification of the difference between the gray levels on the digital image. At least 20 different fields (X400) in the cavernous tissue were examined from each tissue section. The mean percent of smooth muscle cell content was then considered. Statistical analysis. Data are expressed as the mean plus or minus standard deviation. Differences between values were evaluated using analysis of variance followed by the Dunnett test. RESULTS
Table 1 shows mean patient age, intracavernous oxygen tension levels in the flaccid state and oxygen tension levels in response to intracavernous injection of the vasoactive drug. No significant difference was noted in oxygen tension level in the flaccid state among the 3 groups of patients. Ten minutes after prostaglandin E l injection mean oxygen tension level in the control group was 83.8 z 6.8 mm. Hg. This mean value was significantly different (p <0.01) from mean oxygen tension in the venogenic (67.8 ? 8.2) and arteriogenic groups (57.8 2 10.7). The mean percent ofcavernous smooth muscle cells in the control group (43.3 2 3.6%) was si@ficantly different from that in the venogenic (34.1 f- 4.6%) and artenogenic groups (29.3 5 3.4%, p <0.01, table 2). The mean values of cavernous brachial oxygen tension index in the ThR1.E 1. Mean cavernous oxygen tension (mm. Hg) values plus and minus standard deviation in flaccid state and 10 minutes afler injection of 20 pg. prostaglandin E l in the control, venogenic and arteriogenic groups Control -.
Vr;
Arterial Lesion
M w n pt. age (yrs.) 35.4 ? 6.7 47.8 t 10.2 52.2 c 8.3 Oxygen tension at flaccidity 36.2 ? 5.8 35.6 % 6.9 34.1 2 6.5 Oxygen tension after pros- 83.8 ? 6.8 67.8 'c 8.2' 57.8 T 10.7* -. twlandin E l injection * Between control and experimental groups p <0.01.
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TABLE 2. Percent of cavernus s m t h muscle fibers and oxygen tension (mm. H& index ratios: cavernous oxygen tension after prostaglandin E l injection divided by bmchi41 (arterial) oxygen tension % Smooth Oxygen Tension Index Ratio Muacle Fiten, Control 0.87 ? 0.07 43.3 2 3.6 Venous leakage 34.1 2 4.6 0.88 t 0.06 Arterial disease 29.3 2 3.4 0.64 0.09 Values are mean plus and minun standard deviation. Between control and experimental groups p ~ 0 . 0 1 . +_
venogenic (0.68 5 0.06) and arteriogenic groups (0.64 t 0.09) were significantly different (p ~ 0 . 0 1compared ) to that of the psychogenic group (0.87 ? 0.07, table 2). DISCUSSION
Penile erection is a neurovascular phenomenon under psychological control. It involves increased arterial flow and venous resistance, and relaxation of the smooth muscle fibers of the sinusoidal spaces.' Previously it has been shown that the smooth muscle cell represents the structural basis for sinusoidal relaxation, a key factor in penile erection.2 However, others have demonstrated that the cavernous smooth muscle cells can be altered by toxic, neurological or ischemic factors. Cigarette smoking and toxic nicotine particles can block the penile erection response by inhibiting smooth muscle relaxation of the erectile tissue.3.4 Furthermore, in animals nicotine interferes with the erectile response of the drug induced erection, which prevents intracavernous pressure increase after cavernous penile stimulation. On the other hand, the neurological factor that causes erectile dysfunction is demonstrated by excising the penile nerve, leading to the alteration of cavernous tissue cells.5 Recently the ischemic factor in the relaxation and contraction of vascular smooth muscle has been demonstrated.6.11-13 Kim et al showed that physiologically low oxygen tensions could help maintain penile flaccidity by inhibiting nitric oxide production, the novel neurotransmitter for erection.6 Furthermore, insufficiently increased oxygen tension could result in erectile dysfunction. They suggested that reduced oxygen tension could be a limiting factor in nitric oxide mediated relaxation in the penile corpus cavernosum regardless of the normal or pathological state of the nerves and endothelium. This hypothesis could partially explain the role of oxygen tension in the physiology of nitric oxide in relation to the mechanism of penile erection. In addition, others have shown that endothelium-derived relaxing factor and nitric oxide are tightly regulated by oxygen tensi0n.1~ To our knowledge no previous studies have demonstrated the correlation between the alteration of cavernous smooth muscle fibers and low oxygen tension. We noted a significant correlation (p c0.05)between the value of cavernous oxygen tension (mm. Hg) in the flaccid phase and the percent of cavernous smooth muscle fibers (fig. 1).f i r prostaglandin E l injection the erectile response was better when oxygen tension value was higher. Indeed, this observation correlated as well with the increase in the percent of cavernous smooth muscle fibers(p <0.01, fig. 1).Sutficientcavernoussmooth muscle is an important factor in the hemodynamic events of penile erection. At the initiation of erection the smooth muscles of the cavernous sinusoids and arterioles relax, and arterial inflow increases immediately with rapid filling of the sinusoidal spaces. In cases of insufficient cavernous muscle this physiological event is blocked or inhibited. Vardi demonstrated that cavernous oxygen tension was significantly lower after the injection of vasoactive drugs in arteriogenic patients compared to those in the control and venogenic groups, which had similar oxygen tension v a 1 ~ e s . l ~
1738
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS
1
cells.16 Based on our findings this alteration could be due to low cavernous oxygen tension. Surprisingly our impotent patients with pure venous leakage and normal arterial flow had low cavernous oxygen tension compared to psychogenic patients, which could explain the influence of other factors in the alteration of cavernous smooth muscle. For instance, in vens occlusive or ischemic priapism there is a decrease in cavernous oxygen tension leading to increased sickling of the red blood cells, which causes more stasis, ischemia, tissue fibrosis and finally loss of erectile f ~ n c t i 0 n . l ~ To exclude the role of aging in the alteration of cavernous smooth muscle cells we compared the pemnt of cavernoussmooth muscle fibers for the impotent groups in this study to that of potent patients in another study (unpublished data). For the same age group the percent of cavernous muscle cells was higher in potent than impotent patients. Furthermore, in our study the mean percent of cavernous muscle fibers in control patients at age 29 years was 44%, 32 years 47.8%, 36 years 48.7%, 39 years 43.5% and 41 years 42.1%. Biopsies of pa20 30 40 50 tients with cavernovenous leakage at ages 34, 35 and 42 years showed 35.2%, 39% and 32.2% cavernous smooth musC8vomouramodh f l b m (%I cle fibers and those of patients with arterial lesions at ages FIG. 1. Significant relation between cavernous oxygen tension 38,40and 47 years showed 29.6%, 34% and 27% cavernous (P02), before (p <0.05) and 10 minutes &r (p cO.01) intracavern- muscle fiber, respectively. This finding also confirmed the ous injection of 20 c ~ g .prostaglandin E l (PGEI) and percent of reduction of cavernous smooth muscle fiber in impotent pacavernous smooth muscle fibers. tients with vascular disease compared to normal potent patients. The value of the cavernous brachial oxygen tension index in arteriogenic patients was also significantly M e r e n t than CONCLUSIONS that of controls. We noted a significant difference in oxygen To our knowledge we report the first study to demonstrate tension values between the psychogenic and impotent groups that insdicient cavernous oxygen tension could be a possible of patients after the intracavernous injection of 20 Fg. proscause of the alteration of penile smooth muscle fibers in taglandin El. The difference between the 2 studies might be impotent patients. Further studies are needed to explore the due to the difference between the vasoactive drugs injected different causes of alterations in Cavernous muscle as an intracavernously. On the other hand, we have observed a important factor in erectile dyshnction. sirrnificantdifference in the ratio of cavernous brachial index Dr. Haibo Zhang and Mr. Philippe m i r y performed the between the psychogenic and venogenic or arteriogenic groups (p <0.01).This ratio represents the results of cavern- statistical analysis, and Mrs. Isabelle Fayt provided technious oxygen tension after prostaglandin El injection divided cal assistance. by the brachial (arterial)oxygen tension. This ratio may be considered a good screening test in the evaluation of impoREFERENCES tence cases. Furthermore, we detected a high correlation (p 1. Lue, T. F. and Tanagho, E. A.: Physiology of erection and phar<0.01)between the increase in oxygen tension ratio and the macological management of impotence. J. Urol., 137: 829, increase in percent of cavernous smooth muscle fibers (fig. 2). 1987. previously it has been shown that the morphological alter- 2. Krane, R. J., Goldstein, I. and Saenz de Tejada, I.: Impotence. ation of the cavernous ultrastructure in patients with arteNew Engl. J. Med., 321: 1648, 1989. riogenic impotence primarily afYects the smooth muscle 3. Glina. S.. Reichelt. A. C.. LeHo. P. P. and Dos Reis. J. M. S. M.: loo
1
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Cavrmwa rmoolk murcIr flbors (w)
FIG. 2. Significant relation between cavernous-brachial index and percent of cavernous smooth muscle fibers in different groups of patients ( p ~ 0 . 0 1P02, ) oxygen tension.
.
Impact of cigarette smoking on papaverine-induced erection. J. Urol., 1 4 0 523, 1988. 4. Juenemann, R-P., Lue, T. F., Luo, J.-A., Benowitz, N. L., Abozeid, M. and Tanagho, E. A,: The effect of cigarette smoking on penile erection. J. Urol., 138 438, 1987. 5. Paick, J.-S.,Goldsmith, P.C., Batra, A. K., Nunes, L. L., Padula, C. A. and Lue, T. F.: Relationship between venous incompetence and cavernous nerve injury: ultrastructural alteration of cavernous smooth muscle in the neurotomized dog. Int. J. Impotence Res., 3 185, 1991. 6. Kim, N., Vardi, Y., Padma-Nathan, H., Daley, J., Goldstein, I. and Saenz de Tejada, I.: Oxygen tension regulates the nitric oxide pathway. Physiological role in penile erection. J. Clin. Invest., 91: 437, 1993. 7. Burnett, A. L., Lowenstein, C. J.,Bredt, D. S.,Chang, T. S. and Snyder, S. H.: Nitric oxide: a physiologic mediator of penile erection. Science, 257: 401, 1992. 8. Wespes, E., Delcour, C., Struyven, J. and Schulman, C. C.: Pharmacocavernometry-cavernography in impotence. Brit. J. Urol., 5 8 429, 1986. 9. Wespes, E. and Schulman, C. C.: Vascular impotence. World J. Urol., 6 144, 1987. 10. Shapiro, E., Hartanto, V. and Lepor, H.: Anti-desmin vs. antiactin for quantifying the area density of prostate smooth muscle. Prostate, 20: 259, 1992. 11. Kovitz, K. L., Aleskowitch, T. D., Sylvester, J. T. and Flavahan,
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS N. A,: Endothelium-derived contracting and relaxing factors contribute t o hypoxic responses of pulmonary arteries. Amer. J. Physiol. part 2,266 H1139,1993. 12. Shad, P. W.,Farrar, M. A. and Zellers, T. M.: Oxygen modulates endothelium-derivedrelaxing factor production in fetal pulmonary arteries. Amer. J. Physiol., part 2,282 H355, 1992. 13. Johns, R. A., Linden, J. M. and Peach, M. J.: Endotheliumdependent relaxation and cyclic GMP accumulation in rabbit pulmonary artery are selectively impaired by moderate hypoxia. Circ. Res.,65:1508,1989. 14. Rengasamy, A. and Johns, R. A.: Characterization of endothelium-derived relaxing factor/nitric oxide synthase from bovine
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cerebellum and mechanism of modulation by high and low oxygen tensions. J. Pharmacol. Exp. Ther., 259: 310, 1991. 15. Vardi, Y.: Role of oxygen tension in the diagnosis of vasculogenic impotence. In: The Role of Alprostadil in the Diagnosis and Treatment of Erectile Dysfunction. Edited by I. Goldstein and T. F. Lue. Kalamazoo, Michigan: Excerpta Media, pp. 84-94. 1993. 16. Persson, C., Diederichs, W., Lue, T. F.,Yen, T. S. B., Fishman, I. J., McLm, P. H.and Tanagho, E. A: Correlation of altered penile ultrastructure with clinical arterial evaluation. J. Uml., 142: 1462,1989. 17. Stackl, W.:Priapism. Curr. Opin. Urol., 2 453, 1992.
Vol. 154, 1736-1739. November 1995 Printed in U.S.A.
THE JOURNAL OF UR0UX;Y Copyright 0 1995 by AMERICAN U R O ~ I CASS~CLM'ION, AL INC.
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS: RELATION AND FUNCTION AHMED A. SA'ITAR, GEORGES SALPIGIDES, JEAN-JACQUES VANDERHAEGHEN, CLAUDE C. SCHULMAN AND ERIC WESPES" From the Department of Urology, Erasme Hospital and Department of Neuropeptides, Faculty of Medicine, University Clinics of Brussels, Brussels, Belgium
ABSTRACT
Purpose: We studied the effect of intracavernous oxygen tension on the alteration of cavernous smooth muscle fibers in potent and impotent men. Materials and Methods: Intracavernous oxygen tension (mm. Hg) was measured during flaccidity and 10 minutes after intracavernous prostaglandin E l injection in psychogenic control patients, and those with venous leakage and arterial lesions. Cavernous biopsies were performed and the percent of smooth muscle fibers was analyzed objectively using immunohistochemical actin anti-actin staining. Simultaneously brachial oxygen tension (mm. Hg) was measured and the cavernous brachial oxygen tension index was then determined. Results: At flaccidity no significant difference was noted in oxygen tension values among the 3 groups of patients. After prostaglandin E l injection cavernous oxygen tension and the cavernous brachial oxygen tension index in the control group were significantly different (p <0.01)from those of the venogenic and arteriogenic groups (p cO.01).The mean percent of cavernous smooth muscle fibers in the control group was sigdicantly different from those of the venous leakage and arterial lesion groups (p <0.01). There was a good correlation between the percent of cavernous muscle fibers and the value of oxygen tension before (p <0.05)and after prostaglandin E l injection (p CO.01). A similar correlation was noted between cavernous muscle fibers and cavernous brachial oxygen tension index in the different groups of patients (p <0.01). Conclusions: Reduction of the intracavernous smooth muscle fibers in impotent patients could be explained by low intracavernous oxygen tension. KEYWORDS:penis; impotence; blood gas monitoring, transcutaneous; prostaglandins E;muscle, smooth Penile erection results from increased arterial flow, sinusoidal smooth muscle relaxation and decreased venous return.1 Impaired relaxation of the cavernous smooth muscle fibers or alteration in its density represents the structural basis for erectile dysfunction.2 Previously it has been demonstrated in animal models that cavernous muscles can be altered by toxic or neurological factors.= Recently the effect of the ischemic factor or low oxygen on cavernous smooth muscles has been studied. It has been shown that low oxygen tension inhibits the release of the endothelium-derived relaxing factor (nitric oxide), the novel neurotransmitter for erection.6a7 Subsequently this low oxygen tension impaired the relaxation of the cavernous trabecular smooth muscle. However, the causes responsible for cavernous smooth muscle fiber alterations were not demonstrated. To study the effect of oxygen on the alteration of cavernous smooth muscle cells we measured the cavernous oxygen level (oxygen tension in mm. Hg) before and 10 minutes after intracavernous injection of 20 pg. prostaglandin E l in psychogenically impotent patients as a control group and impotent patients presenting with a venous leak or arterial lesion. Results were compared with intracavernous muscular quantity. Cavernous biopsies were analyzed objectively and the percent of smooth muscle fibers was determined.
64 years, mean 47) with venogenic impotence and 10 (age 38
to 63 years, mean 52) with arteriogenic impotence. Clinical evaluation. Patients underwent a complete impotence evaluation, including a detailed sequelae history and
physical examination. They were hospitalized for at least 1 night to evaluate nocturnal penile tumescence using the Rigiscant device. The RigiScan did not show normal nocturnal penile tumescence in the impotent patients. Those with pure venous leakage had a flow rate necessary to maintain an artificial erection of greater than 15 ml. per minute after intracavernous injection of 20 pg. prostaglandin Full blood and hormonal assays were done to exclude endocrinological disease. The bulbocavernous reflex was evaluated to exclude a neurological disorder. The penile brachial index was normal and penile pharmacoangiography did not reveal arterial insuficiency.9 Patients diagnosed as having pure arterial disease had abnormal brachial penile indexes. Pharmacoangiography confirmed arterial lesions. Pharmacocavernosometry-pharmacocavernosography was considered to be normal. Based on these evaluations disease was diagnosed as psychogenic in 5 patients, venogenic in 9 and arteriogenic in 10. Analysis of cavernous and brachial blood gases. Intracavernous blood was obtained by aspiration through a 19 gauge needle and a 2 ml. syringe. Blood gases were immediately MATERIALS A N D METHODS measured using an instrumentation laboratory (pH blood gas A total of 24 patients were enrolled in this study and analyzer). Simultaneously brachial (arterial) blood was obclassified into 3 groups, including 5 patients (age 29 to 41 tained and blood gases were immediately measured as deyears, mean 35.4)with psychogenic impotence, 9 (age 34 to scribed. Injection of 20 pg. prostaglandin E l was done using a 27% gauge needle inserted in the contralateral cavernous body. Ten minutes after injection cavernous blood was obAccepted for publication April 7, 1995. * Requests for re rints: Department of Urology, Erasme Hospital, 808 route de Lenni!, 1070 Brussels, Belgium. .t Dacomed. Minneapolis, Minnesota. 1736
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS
tained as described and blood gases were immediately measwed using the same analyzer. S p c i m e n and tissue processing. All patients underwent avernous biopsy. Specimens were immediately fmed in 10% formalin phosphate buffer solution before parafin embedding. The fixed tissue sections were processed through xylene embedded in paraffin and sectioned at 5 pm. One tissue section was stained with hematoxylin and eosin, and examined by light microscopy to evaluate the cavernous tissue pathologkally. Another tissue section was stained with actin anti-actin for immunohistochemical studies to determine the presence of smooth muscle cells.10 Immunohistochemical staining was done according to the biotin-streptavidin-peroxidase technique. Sections were incubated for 15minutes with 2% (volume-in-volume) hydrogen peroxide solution to block endogenous peroxidase activity and processed for immunohistochemical study. After incubation with 1:20 normal sheep serum the sections were incubated overnight (14 to 18 hours) with mouse anti-human anti-actin antibodies in a dilution of 150. The sections were rinsed and incubated for 30 minutes at room temperature successively with sheep anti-mouse immunoglobulin biotin diluted to 1500 and streptavidin, which was coupled to peroxidase (1:1,000) for 30 minutes. Peroxidase activity was revealed by 10 mg./lO ml. 3.3-diaminobenzidine, pH 7.4, containing 0.01% (volumein-volume) hydrogen peroxide for 5 to 10 minutes at room temperature. Computerized image analysis. Quantitative analysis of smooth muscle fibers in cavernous tissue was determined using an image analyzer system combined with a light microscope equipped with a video camera. Images were interactively discriminated and measurements were obtained from the resulting binary images. The percent of smooth muscle cells for each image results from the quantification of the difference between the gray levels on the digital image. At least 20 different fields (X400) in the cavernous tissue were examined from each tissue section. The mean percent of smooth muscle cell content was then considered. Statistical analysis. Data are expressed as the mean plus or minus standard deviation. Differences between values were evaluated using analysis of variance followed by the Dunnett test. RESULTS
Table 1 shows mean patient age, intracavernous oxygen tension levels in the flaccid state and oxygen tension levels in response to intracavernous injection of the vasoactive drug. No significant difference was noted in oxygen tension level in the flaccid state among the 3 groups of patients. Ten minutes after prostaglandin E l injection mean oxygen tension level in the control group was 83.8 z 6.8 mm. Hg. This mean value was significantly different (p <0.01) from mean oxygen tension in the venogenic (67.8 ? 8.2) and arteriogenic groups (57.8 2 10.7). The mean percent ofcavernous smooth muscle cells in the control group (43.3 2 3.6%) was si@ficantly different from that in the venogenic (34.1 f- 4.6%) and artenogenic groups (29.3 5 3.4%, p <0.01, table 2). The mean values of cavernous brachial oxygen tension index in the ThR1.E 1. Mean cavernous oxygen tension (mm. Hg) values plus and minus standard deviation in flaccid state and 10 minutes afler injection of 20 pg. prostaglandin E l in the control, venogenic and arteriogenic groups Control -.
Vr;
Arterial Lesion
M w n pt. age (yrs.) 35.4 ? 6.7 47.8 t 10.2 52.2 c 8.3 Oxygen tension at flaccidity 36.2 ? 5.8 35.6 % 6.9 34.1 2 6.5 Oxygen tension after pros- 83.8 ? 6.8 67.8 'c 8.2' 57.8 T 10.7* -. twlandin E l injection * Between control and experimental groups p <0.01.
1737
TABLE 2. Percent of cavernus s m t h muscle fibers and oxygen tension (mm. H& index ratios: cavernous oxygen tension after prostaglandin E l injection divided by bmchi41 (arterial) oxygen tension % Smooth Oxygen Tension Index Ratio Muacle Fiten, Control 0.87 ? 0.07 43.3 2 3.6 Venous leakage 34.1 2 4.6 0.88 t 0.06 Arterial disease 29.3 2 3.4 0.64 0.09 Values are mean plus and minun standard deviation. Between control and experimental groups p ~ 0 . 0 1 . +_
venogenic (0.68 5 0.06) and arteriogenic groups (0.64 t 0.09) were significantly different (p ~ 0 . 0 1compared ) to that of the psychogenic group (0.87 ? 0.07, table 2). DISCUSSION
Penile erection is a neurovascular phenomenon under psychological control. It involves increased arterial flow and venous resistance, and relaxation of the smooth muscle fibers of the sinusoidal spaces.' Previously it has been shown that the smooth muscle cell represents the structural basis for sinusoidal relaxation, a key factor in penile erection.2 However, others have demonstrated that the cavernous smooth muscle cells can be altered by toxic, neurological or ischemic factors. Cigarette smoking and toxic nicotine particles can block the penile erection response by inhibiting smooth muscle relaxation of the erectile tissue.3.4 Furthermore, in animals nicotine interferes with the erectile response of the drug induced erection, which prevents intracavernous pressure increase after cavernous penile stimulation. On the other hand, the neurological factor that causes erectile dysfunction is demonstrated by excising the penile nerve, leading to the alteration of cavernous tissue cells.5 Recently the ischemic factor in the relaxation and contraction of vascular smooth muscle has been demonstrated.6.11-13 Kim et al showed that physiologically low oxygen tensions could help maintain penile flaccidity by inhibiting nitric oxide production, the novel neurotransmitter for erection.6 Furthermore, insufficiently increased oxygen tension could result in erectile dysfunction. They suggested that reduced oxygen tension could be a limiting factor in nitric oxide mediated relaxation in the penile corpus cavernosum regardless of the normal or pathological state of the nerves and endothelium. This hypothesis could partially explain the role of oxygen tension in the physiology of nitric oxide in relation to the mechanism of penile erection. In addition, others have shown that endothelium-derived relaxing factor and nitric oxide are tightly regulated by oxygen tensi0n.1~ To our knowledge no previous studies have demonstrated the correlation between the alteration of cavernous smooth muscle fibers and low oxygen tension. We noted a significant correlation (p c0.05)between the value of cavernous oxygen tension (mm. Hg) in the flaccid phase and the percent of cavernous smooth muscle fibers (fig. 1).f i r prostaglandin E l injection the erectile response was better when oxygen tension value was higher. Indeed, this observation correlated as well with the increase in the percent of cavernous smooth muscle fibers(p <0.01, fig. 1).Sutficientcavernoussmooth muscle is an important factor in the hemodynamic events of penile erection. At the initiation of erection the smooth muscles of the cavernous sinusoids and arterioles relax, and arterial inflow increases immediately with rapid filling of the sinusoidal spaces. In cases of insufficient cavernous muscle this physiological event is blocked or inhibited. Vardi demonstrated that cavernous oxygen tension was significantly lower after the injection of vasoactive drugs in arteriogenic patients compared to those in the control and venogenic groups, which had similar oxygen tension v a 1 ~ e s . l ~
1738
CAVERNOUS OXYGEN TENSION AND SMOOTH MUSCLE FIBERS
1
cells.16 Based on our findings this alteration could be due to low cavernous oxygen tension. Surprisingly our impotent patients with pure venous leakage and normal arterial flow had low cavernous oxygen tension compared to psychogenic patients, which could explain the influence of other factors in the alteration of cavernous smooth muscle. For instance, in vens occlusive or ischemic priapism there is a decrease in cavernous oxygen tension leading to increased sickling of the red blood cells, which causes more stasis, ischemia, tissue fibrosis and finally loss of erectile f ~ n c t i 0 n . l ~ To exclude the role of aging in the alteration of cavernous smooth muscle cells we compared the pemnt of cavernoussmooth muscle fibers for the impotent groups in this study to that of potent patients in another study (unpublished data). For the same age group the percent of cavernous muscle cells was higher in potent than impotent patients. Furthermore, in our study the mean percent of cavernous muscle fibers in control patients at age 29 years was 44%, 32 years 47.8%, 36 years 48.7%, 39 years 43.5% and 41 years 42.1%. Biopsies of pa20 30 40 50 tients with cavernovenous leakage at ages 34, 35 and 42 years showed 35.2%, 39% and 32.2% cavernous smooth musC8vomouramodh f l b m (%I cle fibers and those of patients with arterial lesions at ages FIG. 1. Significant relation between cavernous oxygen tension 38,40and 47 years showed 29.6%, 34% and 27% cavernous (P02), before (p <0.05) and 10 minutes &r (p cO.01) intracavern- muscle fiber, respectively. This finding also confirmed the ous injection of 20 c ~ g .prostaglandin E l (PGEI) and percent of reduction of cavernous smooth muscle fiber in impotent pacavernous smooth muscle fibers. tients with vascular disease compared to normal potent patients. The value of the cavernous brachial oxygen tension index in arteriogenic patients was also significantly M e r e n t than CONCLUSIONS that of controls. We noted a significant difference in oxygen To our knowledge we report the first study to demonstrate tension values between the psychogenic and impotent groups that insdicient cavernous oxygen tension could be a possible of patients after the intracavernous injection of 20 Fg. proscause of the alteration of penile smooth muscle fibers in taglandin El. The difference between the 2 studies might be impotent patients. Further studies are needed to explore the due to the difference between the vasoactive drugs injected different causes of alterations in Cavernous muscle as an intracavernously. On the other hand, we have observed a important factor in erectile dyshnction. sirrnificantdifference in the ratio of cavernous brachial index Dr. Haibo Zhang and Mr. Philippe m i r y performed the between the psychogenic and venogenic or arteriogenic groups (p <0.01).This ratio represents the results of cavern- statistical analysis, and Mrs. Isabelle Fayt provided technious oxygen tension after prostaglandin El injection divided cal assistance. by the brachial (arterial)oxygen tension. This ratio may be considered a good screening test in the evaluation of impoREFERENCES tence cases. Furthermore, we detected a high correlation (p 1. Lue, T. F. and Tanagho, E. A.: Physiology of erection and phar<0.01)between the increase in oxygen tension ratio and the macological management of impotence. J. Urol., 137: 829, increase in percent of cavernous smooth muscle fibers (fig. 2). 1987. previously it has been shown that the morphological alter- 2. Krane, R. J., Goldstein, I. and Saenz de Tejada, I.: Impotence. ation of the cavernous ultrastructure in patients with arteNew Engl. J. Med., 321: 1648, 1989. riogenic impotence primarily afYects the smooth muscle 3. Glina. S.. Reichelt. A. C.. LeHo. P. P. and Dos Reis. J. M. S. M.: loo
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FIG. 2. Significant relation between cavernous-brachial index and percent of cavernous smooth muscle fibers in different groups of patients ( p ~ 0 . 0 1P02, ) oxygen tension.
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Impact of cigarette smoking on papaverine-induced erection. J. Urol., 1 4 0 523, 1988. 4. Juenemann, R-P., Lue, T. F., Luo, J.-A., Benowitz, N. L., Abozeid, M. and Tanagho, E. A,: The effect of cigarette smoking on penile erection. J. Urol., 138 438, 1987. 5. Paick, J.-S.,Goldsmith, P.C., Batra, A. K., Nunes, L. L., Padula, C. A. and Lue, T. F.: Relationship between venous incompetence and cavernous nerve injury: ultrastructural alteration of cavernous smooth muscle in the neurotomized dog. Int. J. Impotence Res., 3 185, 1991. 6. Kim, N., Vardi, Y., Padma-Nathan, H., Daley, J., Goldstein, I. and Saenz de Tejada, I.: Oxygen tension regulates the nitric oxide pathway. Physiological role in penile erection. J. Clin. Invest., 91: 437, 1993. 7. Burnett, A. L., Lowenstein, C. J.,Bredt, D. S.,Chang, T. S. and Snyder, S. H.: Nitric oxide: a physiologic mediator of penile erection. Science, 257: 401, 1992. 8. Wespes, E., Delcour, C., Struyven, J. and Schulman, C. C.: Pharmacocavernometry-cavernography in impotence. Brit. J. Urol., 5 8 429, 1986. 9. Wespes, E. and Schulman, C. C.: Vascular impotence. World J. Urol., 6 144, 1987. 10. Shapiro, E., Hartanto, V. and Lepor, H.: Anti-desmin vs. antiactin for quantifying the area density of prostate smooth muscle. Prostate, 20: 259, 1992. 11. Kovitz, K. L., Aleskowitch, T. D., Sylvester, J. T. and Flavahan,
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