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Intracavernous Pressure Responses to Physical and Electrical Stimulation of the Cavernous Nerve in Rats
BASIC SCIENCE
INTRACAVERNOUS PRESSURE RESPONSES TO PHYSICAL AND ELECTRICAL STIMULATION OF THE CAVERNOUS NERVE IN RATS JAMIL REHMAN, GEORGE CHRIST, ARNOLD MELMAN,
AND
JONATHAN FLEISCHMANN
ABSTRACT Objectives. To better define the techniques of nerve-sparing prostate dissection that would result in preservation of erectile function, we characterize the effects of physical pressure on the prostate and cavernous nerve, electrical stimulation of the cavernous nerve, and pharmacologic manipulations on intracavernous pressure (ICP) in normal and diabetic rats. Methods. Fischer-34 rats, both normal and diabetic, underwent dissections that isolated the cavernous bodies and cavernous nerves. Cavernous body pressures were characterized during surgical manipulation, during electrical stimulation of the cavernous nerves, and following papaverine hydrochloride injection. Results. In normal rats, baseline cavernous pressures ranged from 5 to 15 cm H2O (mean 12.29). In diabetic rats, the baseline pressure was significantly lower (3 to 7.5 cm H2O). Lateral nerve displacement caused ICP to rise to approximately 35 cm H2O in normal rats, but only to 20 cm H2O in diabetic rats. Electrostimulation resulted in cavernous pressure increases of 10-fold from baseline in normal rats and sevenfold from baseline in diabetic rats. ICPs were not disturbed appreciably with nerve-sparing dissection techniques. Neurotomy resulted in declines in baseline cavernous pressures in all rats. Electrostimulation of the distal end of a severed nerve resulted in pressure rises to 50% of those observed in rats with intact cavernous nerves. Intracavernous papaverine injection before or after nerve stimulation masked subsequent (expected) pressure changes. Conclusions. A change in cavernous pressure is a sensitive indicator of cavernous nerve manipulation. Both cavernous pressure measurements and electrostimulation of cavernous nerves may aid surgeons during radical prostatectomy. UROLOGY 51: 640–644, 1998. © 1998, Elsevier Science Inc. All rights reserved.
W
alsh and coworkers1,2 popularized the anatomic approach to radical prostatectomy, a technique designed to spare the cavernous nerves and preserve erectile function. Impotence, however, continues to be a postoperative consequence for many patients who undergo this operation, even if the nerves were thought to have been spared. Although vascular injury during prostatectomy contributes to impotence in some patients, unrecognized nerve injury is the probable cause for impotence in the majority of patients. To better define the limits of cavernous nerve manipulations, we explored the utility of intracavernous From the Department of Urology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York Reprint requests: Jonathan Fleischmann, M.D., Albert Einstein College of Medicine, Department of Urology, Weiller Hospital of AECOM, 1825 Eastchester Road, Room 2S 62, Bronx, NY 10461 Submitted: July 14, 1997, accepted (with revisions): October 14, 1997
640
© 1998, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
pressure (ICP) measurements under various surgical and experimental conditions in rats. MATERIAL AND METHODS ANIMALS Two-month-old male Fischer-34 rats (Taconic Farms, Germantown, NY) were fed Purina rodent chow ad libitum and housed individually with a 7 AM to 7 PM light cycle. The animals were divided into three groups: group 1 consisted of control rats (n 5 10); group 2 consisted of diabetic rats (n 5 5); and group 3 consisted of rats who received physical and neurostimulation after intracavernous injection of papaverine hydrochloride (Eli Lilly) (n 5 10). (A total of 10 diabetic rats were included at the outset of this study; 4 died within 3 months of induction of diabetes and 1 died during surgical dissection; therefore, the results of 5 diabetic rats are available for this study.)
INDUCTION OF DIABETES Diabetes mellitus was induced for 3 months in 5 animals by one intraperitoneal injection of streptozotocin (STZ; 35 mg/ kg, Sigma Chemicals) dissolved in citrate buffer (0.1 M citric 0090-4295/98/$19.00 PII S0090-4295(97)00693-6
FIGURE 1. The in vivo rat model used in this study. The rat is anesthetized and lying supine. The arterial line in the left carotid artery is connected to a MacLab data acquisition board via a transducer and transducer amplifier, for continuous monitoring of blood pressure. A right external jugular venous line is used for intravenous fluid transfusion or blood sampling. The prostate has been exposed and the cavernous nerves are seen on the posterolateral surface of the prostate arising from the pelvic ganglion, which is formed by the joining of the hypogastric and pelvic nerves. The two corpora cavernosum have been exposed. A line is inserted into the right corpus cavernosum for continuous monitoring of intracorporal pressure via the MacLab instrumentation and the second line is inserted in the left corpora cavernosum for intracavernous drug injection. Finally, the nerve stimulator probe is placed around the cavernous nerve for current stimulation.
acid 60 mL, 0.2 M Na2HPO4, 40 mL). Rats became hyperglycemic within 24 hours. The extent of diabetes was monitored weekly by urine glucose analysis using a Labstix indicator (Amesdiastix, Miles, Elkhartin), and also confirmed at death by glucose analysis of blood obtained from cardiac puncture (Chemstrip, Boehringer Mannheim, Germany).
SURGICAL TECHNIQUE FOR IN VIVO PRESSURE MONITORING Anesthesia and surgical preparation were performed 12 weeks after the induction of experimental diabetes (the time when documented autonomic neuropathy as well as sensory neuropathy3 is known to be present in diabetic rats). Anesthesia was induced in both age-matched controls and diabetic animals by intraperitoneal injection (35 mg/kg) of sodium pentobarbital (Anpro Pharmaceuticals). Anesthesia was maintained during the course of the experimental protocol (2 to 3 hours) by subsequent injection of pentobarbital (5 to 10 mg/ kg) every 45 to 60 minutes, as required for maintenance of anesthesia.
PLACEMENT OF PRESSURE MONITORING CANNULAE Figure 1 illustrates the experimental procedure. Animals were placed in the supine position and the bladder and prosUROLOGY 51 (4), 1998
tate were exposed through a midline abdominal incision. Prostatic dissection and cavernous nerve isolation was carried out with the rats in a supine position. The inferior hypogastric plexus (ie, the pelvic plexus or major pelvic ganglia), pelvic nerves, and cavernous nerve were identified posterolateral to the prostate on both sides, and stainless-steel bipolar wire electrodes were placed around these structures for electrical stimulation. The penis was degloved, and both corpora cavernosa were exposed by removing part of the overlying ischiocavernous muscle. In order to monitor ICP, a 23-gauge cannula was filled with 250 U/mL of heparin solution, connected to PE-50 tubing (Intramedi, Becton Dickinson), and inserted into the right corpus cavernosum. Another 23-gauge cannula was connected to a 1-mL syringe and inserted into the left corpus cavernosum for intracavernous drug injection. Systemic arterial blood pressure was monitored via a 25-gauge cannula placed into the carotid artery (see Fig. 1). Both pressure lines were connected to a pressure transducer, which was, in turn, connected via a Transducer amplifier (ETH 400, CB Sciences) to a data acquisition board (MacLab/8e, ADI Instruments). Real-time display and recording of pressure measurements was performed on a Macintosh computer (MacLab software V3.4, ADI); see Figure 1 for more details. The pressure transducers and analog/digital (A/D) board were calibrated in cm H2O before each experiment. 641
642
KEY: CN 5 cavernous nerve; ICP 5 intracavernous pressure; IC 5 intracorporal; NC 5 no change observed as the corporal pressure was already higher than 70 cm H2O after IC papaverine and all changes are submerged under this pressure; SE 5 standard error of the mean. * CN stimulation (1 mA) performed on right and left sides (observation multiplied by two times the number of rats).
NC NC 113.54 6 10.83 NC NC 13.18 6 1.392
NC
41.23 6 3.2 0.9 6 0.01 74.81 6 11.13 1.13 6 0.15 19.31 6 3.32
37.88 6 9.38
62.18 6 5.8 6 0.02 2 118.59 6 9.35 1.32 6 0.49 52.51 6 6.37
7.54 6 1.23
FUNCTIONAL STUDIES In normal rats (group 1), baseline cavernous pressure ranged from 5 to 15 cm H2O (mean 12.29) (Table I). In diabetic rats (group 2), the baseline pressure was significantly lower (mean 7.54). Nerve displacement by lateral or posterior movement caused the ICP to rise by approximately 30 cm H2O in control rats; in diabetic rats, the mean increase was 20 cm H2O. Electrostimulation in normal rats (Fig. 2) resulted in intracavernous pressure increases of approximately 10-fold from baseline (;80% of systemic blood pressure), but of
30.31 6 4.31
INDUCTION OF DIABETES There were 5 diabetic rats (urine glucose greater than 1000 mg/dL; blood glucose greater than 400 mg/dL) whose mean weight decreased from 234 to 198 g, consistent with results achieved in previous studies.4 In control animals, the body weight rose an average of 25% during the corresponding time period.
12.29 6 1.01
RESULTS
Group 1 (n 5 10) (control rats) Group 2 (n 5 5) (diabetic rats) Group 3 (n 5 10) (1 IC papaverine)
All statistical analyses were performed using STAT-VIEW 4.5 software (Abacus Concepts, Berkeley, Calif). The ANOVA test was used for comparisons between experimental groups and within each test group. Unless otherwise stated, all cavernous pressure data were expressed as the mean 6 standard error (SE). Comparison between group means was accomplished with a Scheffe´ multiple range test at significance levels of P 5 0.05.
Direct Pressure on CN (Mean 6 SE)
STATISTICAL ANALYSES
Neurostimulation of (intact) (CN)* (Mean 6 SE)
After completion of the neurostimulation protocol, a resting period sufficient to ensure return of basal intracorporal pressure was allowed (;15 to 30 minutes) prior to initiation of the pharmacologic experiments. When basal intracorporal pressure was achieved, papaverine (Eli Lilly) was injected into the left crurum (corpus cavernosum) using a 23-gauge needle attached to a syringe. Four bolus injections of papaverine (maximal injection volume of 60 mL) were administered to each rat at half-log increments ranging from 100 to 3000 mg (ie, 100, 300, 1000, 3000 mg). The change in intracorporal pressure was monitored with each dose, and all pressure responses were allowed to return to baseline before injection of the subsequent dose lasting ;15 to 30 minutes in most cases.
Nerve-Sparing Dissection (Mean 6 SE)
PHARMACOLOGIC PROTOCOL
Stretching/Lateral Displacement of CN
Direct physical pressure was applied by cotton tip swab to both sides of the prostate as well as the cavernous nerve.
TABLE I. Intracavernous pressure: response to dissection and papaverine injection
PHYSICAL MANIPULATION PROTOCOL
Basal ICP (cm H2O) (Mean 6 SE)
Direct electrostimulation of the cavernous nerve was performed with a delicate stainless-steel bipolar hook electrode attached to the multijointed clamp. Each probe was 0.2 mm in diameter; the two poles were 1 mm apart. Monophasic rectangular pulses were delivered by a signal generator (custom made, with a built-in constant current amplifier). Stimulation parameters were as follows: frequency, 20 Hz; pulse width, 0.22 ms; duration, 1 minute; current, 1 mA. Before the experiment, cavernous nerves were cut and neurostimulation of the cut end of the nerve was performed.
Neurotomy (Drop in ICP) (cm H2O) (Mean 6 SE)
Neurostimulation of Distal (Cut) Nerve Ends (Mean 6 SE)
NEUROSTIMULATION PROTOCOL
UROLOGY 51 (4), 1998
servations of the effects of nerve displacement or neurotomy (group 3). Electrostimulation of the distal end of a severed nerve resulted in three- to fivefold intracavernous pressure rise in all animals (50% reduction as compared to intact nerve). When electrostimulation was terminated, pressure declined to 10% of baseline level within 2 minutes and returned to baseline in 5 to 10 minutes in control rats. Intracavernous pressures in diabetic rats declined more rapidly than in controls after termination of electrostimulation. COMMENT
FIGURE 2. Intracavernous pressures: response to electrostimulation of intact cavernous nerve in control rats (n 5 10). Intracavernous pressure in relation to neurostimulation (1NS) with 0.5 to 10 mA of current, 20-Hz pulse, 0.22-second duration, and to withdrawal (2NS) of current.
FIGURE 3. Intracavernous pressures: response to electrostimulation of intact cavernous nerve in diabetic rats (n 5 5). Intracavernous pressure in relation to neurostimulation (1NS) with 0.5 to 10 mA of current, 20-Hz pulse, 0.22-second duration, and to withdrawal (2NS) of current.
only sevenfold from baseline in diabetic rats (Fig. 3). The ICP was not deflected from baseline appreciably using nerve-sparing techniques during periprostatic dissection. Neurotomy resulted in measurable declines (;20% to 30% from baseline) in intracavernous pressures in all rats. Papaverine injections resulted in intracavernous pressure change increases equal to those observed with electrostimulation, but papaverine stimulation of the cavernous body did not enhance sensitivity for obUROLOGY 51 (4), 1998
In 1982, Walsh and Donker1 described the course and relations of the cavernous nerves in stillborn fetuses. The autonomic branches of the pelvic plexus, which lie in the dorsolateral, periprostatic neurovascular bundle, are essential for normal sexual function.5–7 The nerve-sparing radical prostatectomy technique emphasizes the need for direct visualization of these nerves; currently, it cannot be predicted at the time of surgery whether the preserved nerves are functionally normal or if subsequent erectile dysfunction might be a consequence of vascular damage.8 –10 Our investigation sought to characterize the functional consequences of nerve displacement or of nerve injury at the time of surgery in rats. Our data show that the cavernous nerves can be stimulated by tangential or lateral displacements, and that these stimulations have measurable effects on ICP, even in the absence of an observable erection. Moreover, a careful, anatomic dissection to free the cavernous nerves from their tenuous attachments to the dorsolateral surface of the prostate can be performed without demonstrable changes in intracavernous pressure and with full preservation of function. In rats there is a single cavernous nerve, as opposed to man, in whom there are multiple nerves, and anatomy is more complex but functional aspects are the same. In 1863, Eckhard11 described the phenomenon of erections in animals following applications of an electric current to their sacral nerve roots. Subsequent investigators learned to apply the current selectively to specific nerve roots.12,13 Because of the similarities between humans and rats with respect to erections, the rat model has gained wide acceptance.14 –20 In a previous investigation, we showed that nerve-mediated erections, but not exogenous, pharmacologically induced erections, were among the consequences of florid diabetes in rats.20 In the current investigation, diabetic rats consistently showed lower intracavernous pressure increases in response to physical manipulation, pharmacologic stimulation, and electrostimulation of cavernous nerve, and these data 643
support the observations of other investigators.21,22 Quinlan et al.23 showed that the immediate repair of the cavernous nerve following injury restored erectile function in the majority of rats within 4 months of surgery. Similarly, Carrier et al.24 demonstrated the possibility of nerve regeneration following neurotomy. Taken together with our data, these studies suggest that the potential clinical value of electrostimulation and intracavernous pressure monitoring during radical prostatectomy is threefold: first, the specific course and relations of the cavernous nerves can be mapped precisely prior to dissection; second, specific sites of neurotomy can be identified; and third, preoperative erectile function (or dysfunction) can be documented and used to guide postoperative sexual rehabilitation, if needed. These types of clinical investigations are ongoing at our institution. REFERENCES 1. Walsh PC, and Donker PJ: Impotence following radical prostatectomy: insight into etiology and prevention. J Urol 128: 492– 497, 1982. 2. Walsh PC, and Partin AW: Treatment of early stage prostate cancer: radical prostatectomy. Imp Adv Oncol: 211– 223, 1994. 3. Apfel SC, Arezzo JC, Brownlee M, Federoff H, and Kessler JA: Nerve growth factor administration protects against experimental diabetic sensory neuropathy. Brain Res 634: 7–12, 1994. 4. Christ GJ, Valcic M, and Gondre MC: Augmentation in the kinetic characteristics of phenylephrine- and 5-hydroxytryptamine-induced contractions in the isolated rat aorta following eight weeks of STZ-diabetes. Life Sci 55: 807– 814, 1994. 5. Lue TF, Zeineh SJ, Schmidt RA, and Tanagho EA: Neuroanatomy of penile erection: its relevance to iatrogenic impotence. J Urol 131: 273–280, 1984. 6. Lepor H, Gregerman M, Crosby R, Mostofi FK, and Walsh PC: Precise localization of the autonomic nerves from the pelvic plexus to the corpora cavernosa: a detailed anatomical study of the adult male pelvis. J Urol 133: 207–212, 1985. 7. Breza J, Aboseif SR, Orvis BR, Lue TF, and Tanagho EA: Detailed anatomy of penile neurovascular structures: surgical significance. J Urol 141: 437– 443, 1989. 8. Aboseif S, Shinohara K, Breza J, Benard F, and Narayan P: Role of penile vascular injury in erectile dysfunction after radical prostatectomy. Br J Urol 73: 75– 82, 1994. 9. Bahnson RR, and Catalona WJ: Papaverine testing of
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impotent patients following nerve-sparing radical prostatectomy. J Urol 139: 773–774, 1988. 10. Lue TF, Gleason CA, Brock GB, Carroll PR, and Tanagho EA: Intraoperative electrostimulation of the cavernous nerve: technique, results and limitations. J Urol 154: 1426 – 1428, 1995. 11. Eckhard C: Untersuchungen uber die Erektion des penis beim Hunde. Beitr Anat Physiol 3: 123–170, 1863. 12. Langley JN, and Anderson HK: The innervation of the pelvic and adjoining viscera. J Physiol 19: 71–130, 1895. 13. Langley JN: On the regeneration of pre-ganglionic visceral nerve fibers. J Physiol (London) 22: 215–230, 1897. 14. Lesson TL, and Lesson R: The fine structure of the cavernous tissue in the adult rat penis. Invest Urol 3: 144 –154, 1965. 15. Quinlan DM, Nelson RJ, Partin AW, Mostwin JL, and Walsh PC: The rat as a model for the study of penile erection. J Urol 141: 656 – 661, 1989. 16. Dail WG, Walton G, and Olmsted MP: Penile erection in the rat: stimulation of the hypogastric nerve elicits increases in penile pressure after chronic interruption of the sacral parasympathetic outflow. J Auton Nerv Sys 28: 251–257, 1989. 17. Fernandez E, Dail WG, Walton G, and Martinez G: The vasculature of the rat penis: a scanning electron microscopic and histologic study. Am J Anat 192: 307–318, 1991. 18. Chen KK, Chan JY, Chang LS, Chen MT, and Chan JH: Intracavernous pressure as an Experimental index in a rat model for the evaluation of penile erection. J Urol 147: 1124 – 1128, 1992. 19. Martinez-Pineiro L, Brock G, Trigo-Rocha F, Hsu GL, Lue TF, and Tanagho EA: Rat model for the study of penile erection: pharmacologic and electrical stimulation parameters. Eur Urol 25: 62–70, 1994. 20. Rehman J, Chenven E, Brink P, Peterson B, Walcott B, Wen YP, Melman A, and Christ G: Diminished neurogenic but not pharmacological in the 2 to 3 month experimentally diabetic F-344 rat. Am J Physiol 272(Heart Circ Physiol 41): H1960 –H1971, 1997. 21. Steers WD, Mackway-Gerardi AM, Ciamboui J, and DeGroat WC: Alteration in neural pathways to the urinary bladder of the rat in response to streptozotocin-induced diabetes. J Auton Nerv Sys 47: 83–94, 1994. 22. Bemelmans BL, Meuleman EJ, Doesburg WH, Notermans SL, and Debruyne FM: Erectile dysfunction in diabetic men: the neurological factor revisited. J Urol 151: 884 – 889, 1994. 23. Quinlan DM, Nelson RJ, and Walsh PC: Cavernous nerve grafts restore erectile function in denervated rats. J Urol 145: 380 –383, 1991. 24. Carrier S, Zvara P, Nunes L, Kour NW, Rehman J, and Lue TF: Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J Urol 153: 1722–1727, 1995.
UROLOGY 51 (4), 1998
INTRACAVERNOUS PRESSURE RESPONSES TO PHYSICAL AND ELECTRICAL STIMULATION OF THE CAVERNOUS NERVE IN RATS JAMIL REHMAN, GEORGE CHRIST, ARNOLD MELMAN,
AND
JONATHAN FLEISCHMANN
ABSTRACT Objectives. To better define the techniques of nerve-sparing prostate dissection that would result in preservation of erectile function, we characterize the effects of physical pressure on the prostate and cavernous nerve, electrical stimulation of the cavernous nerve, and pharmacologic manipulations on intracavernous pressure (ICP) in normal and diabetic rats. Methods. Fischer-34 rats, both normal and diabetic, underwent dissections that isolated the cavernous bodies and cavernous nerves. Cavernous body pressures were characterized during surgical manipulation, during electrical stimulation of the cavernous nerves, and following papaverine hydrochloride injection. Results. In normal rats, baseline cavernous pressures ranged from 5 to 15 cm H2O (mean 12.29). In diabetic rats, the baseline pressure was significantly lower (3 to 7.5 cm H2O). Lateral nerve displacement caused ICP to rise to approximately 35 cm H2O in normal rats, but only to 20 cm H2O in diabetic rats. Electrostimulation resulted in cavernous pressure increases of 10-fold from baseline in normal rats and sevenfold from baseline in diabetic rats. ICPs were not disturbed appreciably with nerve-sparing dissection techniques. Neurotomy resulted in declines in baseline cavernous pressures in all rats. Electrostimulation of the distal end of a severed nerve resulted in pressure rises to 50% of those observed in rats with intact cavernous nerves. Intracavernous papaverine injection before or after nerve stimulation masked subsequent (expected) pressure changes. Conclusions. A change in cavernous pressure is a sensitive indicator of cavernous nerve manipulation. Both cavernous pressure measurements and electrostimulation of cavernous nerves may aid surgeons during radical prostatectomy. UROLOGY 51: 640–644, 1998. © 1998, Elsevier Science Inc. All rights reserved.
W
alsh and coworkers1,2 popularized the anatomic approach to radical prostatectomy, a technique designed to spare the cavernous nerves and preserve erectile function. Impotence, however, continues to be a postoperative consequence for many patients who undergo this operation, even if the nerves were thought to have been spared. Although vascular injury during prostatectomy contributes to impotence in some patients, unrecognized nerve injury is the probable cause for impotence in the majority of patients. To better define the limits of cavernous nerve manipulations, we explored the utility of intracavernous From the Department of Urology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York Reprint requests: Jonathan Fleischmann, M.D., Albert Einstein College of Medicine, Department of Urology, Weiller Hospital of AECOM, 1825 Eastchester Road, Room 2S 62, Bronx, NY 10461 Submitted: July 14, 1997, accepted (with revisions): October 14, 1997
640
© 1998, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
pressure (ICP) measurements under various surgical and experimental conditions in rats. MATERIAL AND METHODS ANIMALS Two-month-old male Fischer-34 rats (Taconic Farms, Germantown, NY) were fed Purina rodent chow ad libitum and housed individually with a 7 AM to 7 PM light cycle. The animals were divided into three groups: group 1 consisted of control rats (n 5 10); group 2 consisted of diabetic rats (n 5 5); and group 3 consisted of rats who received physical and neurostimulation after intracavernous injection of papaverine hydrochloride (Eli Lilly) (n 5 10). (A total of 10 diabetic rats were included at the outset of this study; 4 died within 3 months of induction of diabetes and 1 died during surgical dissection; therefore, the results of 5 diabetic rats are available for this study.)
INDUCTION OF DIABETES Diabetes mellitus was induced for 3 months in 5 animals by one intraperitoneal injection of streptozotocin (STZ; 35 mg/ kg, Sigma Chemicals) dissolved in citrate buffer (0.1 M citric 0090-4295/98/$19.00 PII S0090-4295(97)00693-6
FIGURE 1. The in vivo rat model used in this study. The rat is anesthetized and lying supine. The arterial line in the left carotid artery is connected to a MacLab data acquisition board via a transducer and transducer amplifier, for continuous monitoring of blood pressure. A right external jugular venous line is used for intravenous fluid transfusion or blood sampling. The prostate has been exposed and the cavernous nerves are seen on the posterolateral surface of the prostate arising from the pelvic ganglion, which is formed by the joining of the hypogastric and pelvic nerves. The two corpora cavernosum have been exposed. A line is inserted into the right corpus cavernosum for continuous monitoring of intracorporal pressure via the MacLab instrumentation and the second line is inserted in the left corpora cavernosum for intracavernous drug injection. Finally, the nerve stimulator probe is placed around the cavernous nerve for current stimulation.
acid 60 mL, 0.2 M Na2HPO4, 40 mL). Rats became hyperglycemic within 24 hours. The extent of diabetes was monitored weekly by urine glucose analysis using a Labstix indicator (Amesdiastix, Miles, Elkhartin), and also confirmed at death by glucose analysis of blood obtained from cardiac puncture (Chemstrip, Boehringer Mannheim, Germany).
SURGICAL TECHNIQUE FOR IN VIVO PRESSURE MONITORING Anesthesia and surgical preparation were performed 12 weeks after the induction of experimental diabetes (the time when documented autonomic neuropathy as well as sensory neuropathy3 is known to be present in diabetic rats). Anesthesia was induced in both age-matched controls and diabetic animals by intraperitoneal injection (35 mg/kg) of sodium pentobarbital (Anpro Pharmaceuticals). Anesthesia was maintained during the course of the experimental protocol (2 to 3 hours) by subsequent injection of pentobarbital (5 to 10 mg/ kg) every 45 to 60 minutes, as required for maintenance of anesthesia.
PLACEMENT OF PRESSURE MONITORING CANNULAE Figure 1 illustrates the experimental procedure. Animals were placed in the supine position and the bladder and prosUROLOGY 51 (4), 1998
tate were exposed through a midline abdominal incision. Prostatic dissection and cavernous nerve isolation was carried out with the rats in a supine position. The inferior hypogastric plexus (ie, the pelvic plexus or major pelvic ganglia), pelvic nerves, and cavernous nerve were identified posterolateral to the prostate on both sides, and stainless-steel bipolar wire electrodes were placed around these structures for electrical stimulation. The penis was degloved, and both corpora cavernosa were exposed by removing part of the overlying ischiocavernous muscle. In order to monitor ICP, a 23-gauge cannula was filled with 250 U/mL of heparin solution, connected to PE-50 tubing (Intramedi, Becton Dickinson), and inserted into the right corpus cavernosum. Another 23-gauge cannula was connected to a 1-mL syringe and inserted into the left corpus cavernosum for intracavernous drug injection. Systemic arterial blood pressure was monitored via a 25-gauge cannula placed into the carotid artery (see Fig. 1). Both pressure lines were connected to a pressure transducer, which was, in turn, connected via a Transducer amplifier (ETH 400, CB Sciences) to a data acquisition board (MacLab/8e, ADI Instruments). Real-time display and recording of pressure measurements was performed on a Macintosh computer (MacLab software V3.4, ADI); see Figure 1 for more details. The pressure transducers and analog/digital (A/D) board were calibrated in cm H2O before each experiment. 641
642
KEY: CN 5 cavernous nerve; ICP 5 intracavernous pressure; IC 5 intracorporal; NC 5 no change observed as the corporal pressure was already higher than 70 cm H2O after IC papaverine and all changes are submerged under this pressure; SE 5 standard error of the mean. * CN stimulation (1 mA) performed on right and left sides (observation multiplied by two times the number of rats).
NC NC 113.54 6 10.83 NC NC 13.18 6 1.392
NC
41.23 6 3.2 0.9 6 0.01 74.81 6 11.13 1.13 6 0.15 19.31 6 3.32
37.88 6 9.38
62.18 6 5.8 6 0.02 2 118.59 6 9.35 1.32 6 0.49 52.51 6 6.37
7.54 6 1.23
FUNCTIONAL STUDIES In normal rats (group 1), baseline cavernous pressure ranged from 5 to 15 cm H2O (mean 12.29) (Table I). In diabetic rats (group 2), the baseline pressure was significantly lower (mean 7.54). Nerve displacement by lateral or posterior movement caused the ICP to rise by approximately 30 cm H2O in control rats; in diabetic rats, the mean increase was 20 cm H2O. Electrostimulation in normal rats (Fig. 2) resulted in intracavernous pressure increases of approximately 10-fold from baseline (;80% of systemic blood pressure), but of
30.31 6 4.31
INDUCTION OF DIABETES There were 5 diabetic rats (urine glucose greater than 1000 mg/dL; blood glucose greater than 400 mg/dL) whose mean weight decreased from 234 to 198 g, consistent with results achieved in previous studies.4 In control animals, the body weight rose an average of 25% during the corresponding time period.
12.29 6 1.01
RESULTS
Group 1 (n 5 10) (control rats) Group 2 (n 5 5) (diabetic rats) Group 3 (n 5 10) (1 IC papaverine)
All statistical analyses were performed using STAT-VIEW 4.5 software (Abacus Concepts, Berkeley, Calif). The ANOVA test was used for comparisons between experimental groups and within each test group. Unless otherwise stated, all cavernous pressure data were expressed as the mean 6 standard error (SE). Comparison between group means was accomplished with a Scheffe´ multiple range test at significance levels of P 5 0.05.
Direct Pressure on CN (Mean 6 SE)
STATISTICAL ANALYSES
Neurostimulation of (intact) (CN)* (Mean 6 SE)
After completion of the neurostimulation protocol, a resting period sufficient to ensure return of basal intracorporal pressure was allowed (;15 to 30 minutes) prior to initiation of the pharmacologic experiments. When basal intracorporal pressure was achieved, papaverine (Eli Lilly) was injected into the left crurum (corpus cavernosum) using a 23-gauge needle attached to a syringe. Four bolus injections of papaverine (maximal injection volume of 60 mL) were administered to each rat at half-log increments ranging from 100 to 3000 mg (ie, 100, 300, 1000, 3000 mg). The change in intracorporal pressure was monitored with each dose, and all pressure responses were allowed to return to baseline before injection of the subsequent dose lasting ;15 to 30 minutes in most cases.
Nerve-Sparing Dissection (Mean 6 SE)
PHARMACOLOGIC PROTOCOL
Stretching/Lateral Displacement of CN
Direct physical pressure was applied by cotton tip swab to both sides of the prostate as well as the cavernous nerve.
TABLE I. Intracavernous pressure: response to dissection and papaverine injection
PHYSICAL MANIPULATION PROTOCOL
Basal ICP (cm H2O) (Mean 6 SE)
Direct electrostimulation of the cavernous nerve was performed with a delicate stainless-steel bipolar hook electrode attached to the multijointed clamp. Each probe was 0.2 mm in diameter; the two poles were 1 mm apart. Monophasic rectangular pulses were delivered by a signal generator (custom made, with a built-in constant current amplifier). Stimulation parameters were as follows: frequency, 20 Hz; pulse width, 0.22 ms; duration, 1 minute; current, 1 mA. Before the experiment, cavernous nerves were cut and neurostimulation of the cut end of the nerve was performed.
Neurotomy (Drop in ICP) (cm H2O) (Mean 6 SE)
Neurostimulation of Distal (Cut) Nerve Ends (Mean 6 SE)
NEUROSTIMULATION PROTOCOL
UROLOGY 51 (4), 1998
servations of the effects of nerve displacement or neurotomy (group 3). Electrostimulation of the distal end of a severed nerve resulted in three- to fivefold intracavernous pressure rise in all animals (50% reduction as compared to intact nerve). When electrostimulation was terminated, pressure declined to 10% of baseline level within 2 minutes and returned to baseline in 5 to 10 minutes in control rats. Intracavernous pressures in diabetic rats declined more rapidly than in controls after termination of electrostimulation. COMMENT
FIGURE 2. Intracavernous pressures: response to electrostimulation of intact cavernous nerve in control rats (n 5 10). Intracavernous pressure in relation to neurostimulation (1NS) with 0.5 to 10 mA of current, 20-Hz pulse, 0.22-second duration, and to withdrawal (2NS) of current.
FIGURE 3. Intracavernous pressures: response to electrostimulation of intact cavernous nerve in diabetic rats (n 5 5). Intracavernous pressure in relation to neurostimulation (1NS) with 0.5 to 10 mA of current, 20-Hz pulse, 0.22-second duration, and to withdrawal (2NS) of current.
only sevenfold from baseline in diabetic rats (Fig. 3). The ICP was not deflected from baseline appreciably using nerve-sparing techniques during periprostatic dissection. Neurotomy resulted in measurable declines (;20% to 30% from baseline) in intracavernous pressures in all rats. Papaverine injections resulted in intracavernous pressure change increases equal to those observed with electrostimulation, but papaverine stimulation of the cavernous body did not enhance sensitivity for obUROLOGY 51 (4), 1998
In 1982, Walsh and Donker1 described the course and relations of the cavernous nerves in stillborn fetuses. The autonomic branches of the pelvic plexus, which lie in the dorsolateral, periprostatic neurovascular bundle, are essential for normal sexual function.5–7 The nerve-sparing radical prostatectomy technique emphasizes the need for direct visualization of these nerves; currently, it cannot be predicted at the time of surgery whether the preserved nerves are functionally normal or if subsequent erectile dysfunction might be a consequence of vascular damage.8 –10 Our investigation sought to characterize the functional consequences of nerve displacement or of nerve injury at the time of surgery in rats. Our data show that the cavernous nerves can be stimulated by tangential or lateral displacements, and that these stimulations have measurable effects on ICP, even in the absence of an observable erection. Moreover, a careful, anatomic dissection to free the cavernous nerves from their tenuous attachments to the dorsolateral surface of the prostate can be performed without demonstrable changes in intracavernous pressure and with full preservation of function. In rats there is a single cavernous nerve, as opposed to man, in whom there are multiple nerves, and anatomy is more complex but functional aspects are the same. In 1863, Eckhard11 described the phenomenon of erections in animals following applications of an electric current to their sacral nerve roots. Subsequent investigators learned to apply the current selectively to specific nerve roots.12,13 Because of the similarities between humans and rats with respect to erections, the rat model has gained wide acceptance.14 –20 In a previous investigation, we showed that nerve-mediated erections, but not exogenous, pharmacologically induced erections, were among the consequences of florid diabetes in rats.20 In the current investigation, diabetic rats consistently showed lower intracavernous pressure increases in response to physical manipulation, pharmacologic stimulation, and electrostimulation of cavernous nerve, and these data 643
support the observations of other investigators.21,22 Quinlan et al.23 showed that the immediate repair of the cavernous nerve following injury restored erectile function in the majority of rats within 4 months of surgery. Similarly, Carrier et al.24 demonstrated the possibility of nerve regeneration following neurotomy. Taken together with our data, these studies suggest that the potential clinical value of electrostimulation and intracavernous pressure monitoring during radical prostatectomy is threefold: first, the specific course and relations of the cavernous nerves can be mapped precisely prior to dissection; second, specific sites of neurotomy can be identified; and third, preoperative erectile function (or dysfunction) can be documented and used to guide postoperative sexual rehabilitation, if needed. These types of clinical investigations are ongoing at our institution. REFERENCES 1. Walsh PC, and Donker PJ: Impotence following radical prostatectomy: insight into etiology and prevention. J Urol 128: 492– 497, 1982. 2. Walsh PC, and Partin AW: Treatment of early stage prostate cancer: radical prostatectomy. Imp Adv Oncol: 211– 223, 1994. 3. Apfel SC, Arezzo JC, Brownlee M, Federoff H, and Kessler JA: Nerve growth factor administration protects against experimental diabetic sensory neuropathy. Brain Res 634: 7–12, 1994. 4. Christ GJ, Valcic M, and Gondre MC: Augmentation in the kinetic characteristics of phenylephrine- and 5-hydroxytryptamine-induced contractions in the isolated rat aorta following eight weeks of STZ-diabetes. Life Sci 55: 807– 814, 1994. 5. Lue TF, Zeineh SJ, Schmidt RA, and Tanagho EA: Neuroanatomy of penile erection: its relevance to iatrogenic impotence. J Urol 131: 273–280, 1984. 6. Lepor H, Gregerman M, Crosby R, Mostofi FK, and Walsh PC: Precise localization of the autonomic nerves from the pelvic plexus to the corpora cavernosa: a detailed anatomical study of the adult male pelvis. J Urol 133: 207–212, 1985. 7. Breza J, Aboseif SR, Orvis BR, Lue TF, and Tanagho EA: Detailed anatomy of penile neurovascular structures: surgical significance. J Urol 141: 437– 443, 1989. 8. Aboseif S, Shinohara K, Breza J, Benard F, and Narayan P: Role of penile vascular injury in erectile dysfunction after radical prostatectomy. Br J Urol 73: 75– 82, 1994. 9. Bahnson RR, and Catalona WJ: Papaverine testing of
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impotent patients following nerve-sparing radical prostatectomy. J Urol 139: 773–774, 1988. 10. Lue TF, Gleason CA, Brock GB, Carroll PR, and Tanagho EA: Intraoperative electrostimulation of the cavernous nerve: technique, results and limitations. J Urol 154: 1426 – 1428, 1995. 11. Eckhard C: Untersuchungen uber die Erektion des penis beim Hunde. Beitr Anat Physiol 3: 123–170, 1863. 12. Langley JN, and Anderson HK: The innervation of the pelvic and adjoining viscera. J Physiol 19: 71–130, 1895. 13. Langley JN: On the regeneration of pre-ganglionic visceral nerve fibers. J Physiol (London) 22: 215–230, 1897. 14. Lesson TL, and Lesson R: The fine structure of the cavernous tissue in the adult rat penis. Invest Urol 3: 144 –154, 1965. 15. Quinlan DM, Nelson RJ, Partin AW, Mostwin JL, and Walsh PC: The rat as a model for the study of penile erection. J Urol 141: 656 – 661, 1989. 16. Dail WG, Walton G, and Olmsted MP: Penile erection in the rat: stimulation of the hypogastric nerve elicits increases in penile pressure after chronic interruption of the sacral parasympathetic outflow. J Auton Nerv Sys 28: 251–257, 1989. 17. Fernandez E, Dail WG, Walton G, and Martinez G: The vasculature of the rat penis: a scanning electron microscopic and histologic study. Am J Anat 192: 307–318, 1991. 18. Chen KK, Chan JY, Chang LS, Chen MT, and Chan JH: Intracavernous pressure as an Experimental index in a rat model for the evaluation of penile erection. J Urol 147: 1124 – 1128, 1992. 19. Martinez-Pineiro L, Brock G, Trigo-Rocha F, Hsu GL, Lue TF, and Tanagho EA: Rat model for the study of penile erection: pharmacologic and electrical stimulation parameters. Eur Urol 25: 62–70, 1994. 20. Rehman J, Chenven E, Brink P, Peterson B, Walcott B, Wen YP, Melman A, and Christ G: Diminished neurogenic but not pharmacological in the 2 to 3 month experimentally diabetic F-344 rat. Am J Physiol 272(Heart Circ Physiol 41): H1960 –H1971, 1997. 21. Steers WD, Mackway-Gerardi AM, Ciamboui J, and DeGroat WC: Alteration in neural pathways to the urinary bladder of the rat in response to streptozotocin-induced diabetes. J Auton Nerv Sys 47: 83–94, 1994. 22. Bemelmans BL, Meuleman EJ, Doesburg WH, Notermans SL, and Debruyne FM: Erectile dysfunction in diabetic men: the neurological factor revisited. J Urol 151: 884 – 889, 1994. 23. Quinlan DM, Nelson RJ, and Walsh PC: Cavernous nerve grafts restore erectile function in denervated rats. J Urol 145: 380 –383, 1991. 24. Carrier S, Zvara P, Nunes L, Kour NW, Rehman J, and Lue TF: Regeneration of nitric oxide synthase-containing nerves after cavernous nerve neurotomy in the rat. J Urol 153: 1722–1727, 1995.
UROLOGY 51 (4), 1998