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Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats
Brain Research 836 Ž1999. 19–30 www.elsevier.comrlocaterbres
Research report
Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats Warren M. Grill ) , Narendra Bhadra, Baoqing Wang Department of Biomedical Engineering, Applied Neural Control Laboratory, C.B. Bolton Building, Room 3480, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106-4912, USA Accepted 4 May 1999
Abstract Experiments were conducted to measure the bladder and urethral pressures evoked by intraspinal microstimulation of the sacral segments ŽS1–S2. in neurologically intact, chloralose anesthetized adult male cats. The bladder pressure was measured with a superpubic catheter and the urethral pressure was measured simultaneously at the level of the urethral sphincter and at the level of the penis using a two-element micromanometer. Intraspinal stimuli Žtypically 1 s, 20 Hz, 100 mA, 100 ms. were applied with activated iridium microwire electrodes in ipsilateral segments and intersegmental boundaries with a 250 mm mediolateral resolution and a 200 mm dorsoventral resolution. Increases in bladder pressures were generated by microstimulation in the intermediolateral region, in the lateral and ventrolateral ventral horn, and around the central canal. Simultaneous increases in urethral pressure were evoked by microstimulation in the ventrolateral ventral horn, but not at the other locations. Small reductions in urethral pressure Ž- 10 cm H 2 O. were evoked at locations in the intermediate laminae and around the central canal. The magnitude of these pressure reductions was weakly dependent on the stimulus parameters. Stimulation around the central canal produced bladder contractions with either no change or a reduction in urethral pressure and voiding of small amounts of fluid. These results demonstrate that regions are present in the spinal intact anesthetized cat where microstimulation generates selective contraction of the bladder without increases in urethral pressure and that regions are present where microstimulation generates small reductions in urethral pressure. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micturition; Genitourinary; Parasympathetic; Electrical stimulation; Neural prosthesis
1. Introduction The genitourinary system is a complex of smooth and striated muscles controlled by somatic and autonomic innervation, including both parasympathetic and sympathetic components w12x. Control of storage and release of urine, the primary functions of the genitourinary system, includes supraspinal, spinal, and ganglionic elements. It has been demonstrated in animals that the normal cycle of micturition involves a spinal–pontine–spinal reflex loop in which descending commands orchestrate contraction of the smooth muscle detrusor and relaxation of periurethral striated musculature w21,25x. Lesion studies w26,40x and a recent imaging study w4x support a role for the dorsolateral pons in control of micturition in the human. ) Corresponding [email protected]
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Spinal cord injury disrupts these reflex loops and leads to changes in the functioning of the system, including an inability to void, hyperreflexia, and bladder–sphincter dyssynergia, which can lead to serious secondary complications including infection, upper urinary tract damage, and hydronephrosis w2x. Electrical activation of intact neuronal structures below the level of the lesion has been investigated for restoration of micturition in persons with spinal cord injury w10,38x. Initial efforts to electrically evacuate the bladder were largely unsuccessful due to concomitant activation of the periurethral striated musculature both by the electrical stimulus and by local reflex actions w41x. A number of methods including pudendal neurectomy, sphincterotomy, and direct bladder stimulation have been attempted to avoid sphincter activation. The most successful to date has been intermittent stimulation of the sacral ventral roots, in which urine flows during the inter-burst interval, combined with sacral dorsal rhizotomy
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 5 8 1 - 4
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w8x. Another method that met with mixed success was direct activation of the ‘‘micturition center’’ in the spinal cord using penetrating microelectrodes w32,33x. Although anatomical and physiological studies in animals have not demonstrated the existence of a spinal micturition center, it may be possible to activate spinal neurons to produce physiological voiding in individuals with spinal cord injury. Importantly, the early human studies demonstrated the feasibility of chronic intraspinal microstimulation. The possibility of restoring bladder function through intraspinal microstimulation holds promise and has not been systematically investigated. Furthermore, the physiological roles of different groups of spinal neurons in control of micturition have yet to be completely understood w13,14,23,29x. The objective of the experiments reported here was to measure the pressures produced in the genitourinary system by microstimulation of the sacral spinal cord. The goal was to determine whether regions could be identified where microstimulation generated selective contraction of the bladder, and whether regions could be identified where microstimulation generated reductions in urethral pressure. Experiments were conducted in spinal-intact animals and these results serve as a baseline against which responses generated by microstimulation in chronic spinal model can be evaluated. Preliminary results have been reported in abstract form w18x.
2. Materials and methods 2.1. Experimental preparation Acute experiments were conducted on adult, sexually intact, male cats Ž2.8–4.5 kg.. All animal care and experimental procedures were according to NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Animals were sedated with xylazine ŽRompun, 2.0 mgrkg, s.q.. and anesthetized with ketamine HCl ŽKetaset, 15 mgrkg, i.m., supplemented as required during surgical preparation.. A venous catheter was inserted in the cephalic vein, and anesthesia maintained with a-chloralose ŽSigma, 60 mgr kg, i.v., supplemented at 15 mgrkg.. A midline abdominal incision was made to expose the bladder, the ureters were ligated, transected proximal to the ligation, and drained externally. The bladder was cannulated with a PE tube introduced with a hypodermic needle and secured with a purse string suture. A vent tube, open to atmosphere, was placed adjacent to the bladder, and the abdominal incision was closed in layers. A laminectomy was made to expose the sacral spinal cord and sacral spinal roots. The animal was mounted in a frame with pins at the hips, the head in a headholder, and a vertebral clamp at L4. Body temperature was maintained between 378 and 398C with a thermostatically controlled heat lamp, warm 0.9% saline with 8.4 mgrcm3 sodium bicarbonate and 5% dextrose added was
administered i.v. Ž; 20 cm3rh., respiration was maintained with a respirator, and the electrocardiogram was monitored throughout the experiment. Dexamethasone Ž2 mgrkg, i.v.. was administered at the completion of the laminectomy and every 6 h thereafter for the duration of the experiment. 2.2. Recording and stimulation Bladder pressure was measured using a solid state pressure transducer connected to the superpubic PE catheter ŽDeltran DPT-100, Utah Medical Products, Midvale, UT.. Bladder volume was maintained at a level Ž5–10 cm3 . that was below the threshold to generate reflex contractions of the bladder, and thus all measurements were made during a low volume, low pressure continence phase of the micturition cycle. Urethral pressures were measured using a custom two-channel micromanometer which had two directionally oriented solid state pressure transducers, spaced 3 cm apart and circumferentially offset by 1808, mounted in a silicone rubber catheter 1.4 mm in diameter ŽModel CTrS2, MMI, Hackensack, NJ.. The catheter was used to measure simultaneously the pressure in the sphincteric and penile portions of the urethra. The catheter was positioned by slowly withdrawing it along the length of the urethra to find the location of maximum pressure. The pressure at this location also exhibited spontaneous fluctuations indicative of the activity of the external urethral sphincter and lay approximately 6 cm from the external meatus w46x. The second sensor was displaced 3 cm distally and lay within the penile urethra. In two experiments, urethral pressures were measured with a fluid-filled PE tubing catheter Žo.d.s 0.96 mm., with a sealed tip and a sidehole of diameter ; 0.8 mm, connected to a solid state pressure transducer. The pressure signals were amplified, low pass filtered Žcut-off frequencys 30 Hz., and continuously recorded on a strip chart recorder. Pressures evoked by stimulation were also sampled by computer Žsampling frequencys 100 Hz.. The dura was opened and the individual dorsal and ventral sacral roots were isolated and stimulated using a hook electrode and constant current stimulator Ž1 s, 20 Hz, 100 ms, 1 mA.. The root that produced the largest bladder pressure was presumed to be S2. This identification was tested by stimulation of the more rostral root, which generated much larger urethral pressures, and confirmed by post-mortem dissection to identify the root exits from the spinal column. After root identification, the cord was covered with warm mineral oil and mapping by microstimulation commenced. Vertical, dorsal-to-ventral penetrations Ždepth increments 100–400 mm, typically 200 mm. were made at multiple mediolateral locations between the midline and the lateral border Ždistance between parallel tracks was typically 250 mm. in the rostrocaudal middle of the S1 Ž n s 17 penetrations across two experiments. and S2 Ž n s 55 penetrations across eight experiments. seg-
W.M. Grill et al.r Brain Research 836 (1999) 19–30
ments, and at intersegmental boundaries between S1 and S2 Ž n s 16 penetrations across two experiments. and S2 and S3 Ž n s 6 penetrations in one experiment.. Activated iridium wire microelectrodes Ž50 mm diameter. with an exposed electrochemically determined surface area of ; 225 mm2 , a 1–3 mm tip, and insulated with Epoxylite were
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used for most penetrations ŽIS300, Huntington Medical Research Institutes, Pasadena, CA., although some 75 mm parylene-insulated iridium wire electrodes with an exposed geometric surface area of 250 mm2 and a 1–2 mm tip were also used ŽIR3PT100, MicroProbe, Clarksburg, MD.. Stimuli were charge balanced biphasic pulses with a ca-
Fig. 1. Bladder and urethral pressures evoked by intraspinal microstimulation of the rostral S2 intermediolateral region and lateral ventral horn. ŽA. Pressures evoked in the bladder and proximal urethra Ž; 6 cm from the external meatus. by intraspinal microstimulation with a 1 s 20 Hz train of 100 ms pulses at the indicated amplitudes. The ‘‘peak of slow component’’ points indicate urethral pressure increases attributable to transmitted pressures from the bladder. ŽB, C. Individual bladder and urethral pressure responses evoked by microstimulation at depths of 2400 mm ŽB. or 3400 mm ŽC. with a 1 s, 20 Hz train of 100 ms, 200 mA pulses. ŽD. Tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the location of the penetration and the locations where the individual responses were evoked ŽB, C..
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thodic phase amplitude of 10–200 mA and duration of 50–400 ms applied as 1 s to 10 s continuous and intermittent trains with a frequency between 2 Hz and 100 Hz. The amplitude of the anodic phase was limited to 100 mA and the duration was set automatically by the stimulator to balance the charge injected in the primary phase. The
standard mapping stimulus was a 1 s train of 100 mA, 100 ms, 20 Hz pulses. At the conclusion of the experiment the spinal cord was fixed in situ by immersion in 10% buffered formalin and the animal was killed by an overdose of sodium pentobarbital. The spinal segments of interest were excised and
Fig. 2. Bladder and urethral pressures evoked by intraspinal microstimulation of the caudal S2 intermediolateral region and lateral ventral horn. ŽA. Pressures evoked in the bladder, proximal urethra, and distal urethra by microstimulation along a penetration through the S2 intermediolateral region. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽB. Traces of the pressures evoked in the bladder ŽPbl., proximal urethra ŽPup, ; 6 cm from external meatus., and distal urethra ŽPud, ; 3 cm from external meatus. by microstimulation at 2200 mm Ža. and 2400 mm Žb. below the surface. Stimulus was a 1 s 20 Hz train of 100 ms, 100 mA pulses. ŽC. Influence of stimulus parameters on the pressures evoked in the bladder ŽPbl., proximal urethra ŽPup., and distal urethra ŽPud. by microstimulation in the S2 spinal cord. The stimulus was located at 1500 mm microns below the surface in the dorsal aspect of the intermediolateral region. ŽD. A tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the location of the penetration and the locations where the individual responses were evoked ŽBa, Bb, C..
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placed in refrigerated 10% buffered formalin. At the completion of mapping a particular transverse segment in some experiments, the electrode was moved contralateral to the region mapped, inserted to a known depth, and cut-off. These electrodes remained in the cord throughout fixation and blocking to identify the rostrocaudal location of the penetrations. Spinal cord sections were blocked, microscopic images were digitized, and outlines of the white matter and gray matter were made using a computer-based image analysis system. Bladder pressures were measured as the peak of the evoked pressure with respect to the baseline pressure in the bladder, the latter averaged over the 1 s interval preceding the stimulus. Urethral pressures were measured as the mean of the pressure evoked during the stimulus train with respect to the baseline pressure in the urethra averaged over the 1 s interval preceding the stimulus. Thus, all pressures are reported as changes relative to the pre-stimulus baseline value, rather than as absolute values. The baseline pressures in the bladder varied from 1 to 21 cm H 2 O and exhibited little variation within an experiment Žaverage range s 5 cm H 2 O.. The baseline pressures in the proximal urethra varied from 9 to 30 cm H 2 O and had greater variation during the experiment Žaverage range s 13 cm H 2 O. than the baseline bladder pressure.
3. Results Genitourinary responses to microstimulation of the sacral spinal cord were assessed by measuring pressure changes in the bladder, proximal Žsphincteric. urethra, and distal Žpenile. urethra. Most penetrations were made in the region of the intermediolateral cell column of the S2 segment, and results are presented based on 94 penetration across eight experiments in which the average number of penetrations in the sacral segments was 13 and the range was 3–22. In some experiments, the hindlimb motor responses evoked by intraspinal stimulation in the lumbar segments were also measured, but these data will be reported separately. The 1-s 20-Hz mapping stimulus produced a monophasic increase in bladder pressure with a time to peak of 2.0–2.5 s, followed by a slow decay to baseline ŽFigs. 1B,C and 2B,C.. Bladder pressures were quantified by measuring the peak amplitude of this pressure increase relative to the baseline pressure preceding the stimulus. Increases ŽFigs. 1C and 2B. or decreases ŽFig. 2C. in urethral pressure were generated with short latency in both the region of the sphincter and in the penis. Urethral pressures were quantified by measuring the average pressure during the stimulus train, relative to the baseline pressure preceding the stimulus. As little striated muscle is present along the penile urethra w46x, the rapid pressure changes in the penile urethra were presumed to be due to activation of the ischiocavernosus and bulbocavernosus
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muscles at the base of the penis. This presumption was supported by the observation that penile pressure increases were correlated with twitching of the penis. 3.1. Segmental distribution of pressure responses Bladder and urethral pressures were evoked by microstimulation at locations spread throughout the sacral segments. The magnitude and characteristics of the pressure responses varied according to the location ŽTable 1., magnitude, and pattern of the stimulus. Larger bladder pressures were evoked by microstimulation within the S2 segment than by microstimulation within the S1 segment, although the difference was not significant. This was consistent with the higher bladder pressures evoked by stimulation of the S2 ventral than the S1 ventral root Ž p 0.0001, t-test of the null hypothesis that the means pressures were the same from S1 and S2 stimulation.. Conversely, larger urethral pressures at the level of the urethral sphincter were evoked by microstimulation within the S1 segment than within the S2 segment, although the difference was not significant. This also was consistent with the higher urethral pressures evoked by stimulation of the S1 ventral root than the S2 ventral root, although this difference was also not significant Ž p s 0.0861, t-test of the null hypothesis that the means pressures were the same from S1 and S2 stimulation.. Thus, the magnitudes of the bladder and urethral pressures evoked by intraspinal microstimulation differed across spinal segments and were correlated with the pressures evoked by spinal root stimulation. 3.2. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the intermediolateral region The amplitude and characteristics of the pressure responses evoked by microstimulation were dependent on the dorsoventral as well as the mediolateral location of the stimulus. An example of an electrode penetration traversing the intermediolateral region and lateral ventral horn of the S2 segment is shown in Fig. 1. Bladder pressures greater than 15 cm H 2 O were consistently evoked by microstimulation in the intermediolateral region and in the
Table 1 Bladder and urethral pressures evoked by intraspinal microstimulation and spinal root stimulation Entries are mean"S.D. Žcm H 2 O. Ž n.. Bladder a
Intraspinal Dorsal root b Ventral root b a b
Urethra
S1
S2
S1
S2
21"17 Ž3. 8"7 Ž6. 9"7 Ž7.
28"9 Ž8. 22"21 Ž6. 43"11 Ž7.
94"73 Ž3. 40"46 Ž6. 66"52 Ž7.
75"77 Ž8. 13"10 Ž6. 20"29 Ž7.
Maximum pressures evoked by 1 s, 20 Hz, 100 mA, 100 ms stimulus. Maximum pressures evoked by 1 s, 20 Hz, 1 mA, 100 ms stimulus.
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W.M. Grill et al.r Brain Research 836 (1999) 19–30
lateral and ventrolateral aspects of the ventral horn. No activation of periurethral striated muscle was evoked by microstimulation in the intermediolateral region or the lateral ventral horn. Microstimulation in the intermediolateral region evoked a slow increase in urethral pressure at the level of the urethral sphincter following stimulation ŽFig. 1B.. This slow, long-latency response was the result of transmitted pressure from the bladder via fluid flow from the bladder to the urethra. Generation of large bladder pressures without increases in urethral pressure by microstimulation in the intermediolateral region and the lateral ventral horn can also be seen in Fig. 4C and D, respectively, as well as in Fig. 2. Microstimulation at locations in the ventrolateral aspect of the ventral horn generated large bladder pressures, but also generated increases in urethral pressure. As shown in Fig. 1C, there was a short-latency increase in urethral pressure which subsided on termination of the stimulus, followed by a slow, long-latency pressure increase transmitted from the bladder. A similar distinction is illustrated in Fig. 2. In this case the baseline pressure proximal to the sensor was sufficient to prevent transmitted pressure from being measured. However, there was a clear distinction between areas in the lateral ventral horn that generated selective activation of the bladder ŽFig. 2Ba., and areas in the ventral aspect of the ventral horn that generated coactivation of the bladder and periurethral striated muscle ŽFig. 2Bb.. These data demonstrate that microstimulation in the area of the intermediolateral region and the lateral ventral horn generated large bladder pressures Ž30–40 cm H 2 O., without co-activation of periurethral musculature. Microstimulation in the ventral aspect of the ventral horn generated large increases in bladder pressure accompanied by large increases in urethral pressure, both at the level of the urethral sphincter and in the penile urethra. Microstimulation in the dorsal aspect of the intermediolateral region produced increases in bladder pressure coupled with small reductions in urethral pressure ŽFig. 2.. Stimulus locations that generated a reduction in urethral pressure were of particular interest, as a drop in urethral pressure accompanies natural micturition. The mapping stimulus generated reductions in pressure of less than 10 cm H 2 O and the influence of stimulus parameters was explored in an attempt to augment these pressure reductions ŽFig. 2C.. In this example, the mapping stimulus generated a small reduction in pressure the proximal urethra, and no change in pressure in the distal urethra.
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Increasing the stimulus amplitude resulted in an increase in the evoked bladder pressure, but did not augment the reduction in urethral pressure. The magnitude of the pressure reduction increased as the stimulus frequency was increased, but reached a plateau at frequencies greater than 40 Hz. Applying longer trains at either 100% or 50% duty cycle did not further augment the pressure reduction. These results show that the stimulus evoked reductions in urethral pressure were only weakly dependent on the stimulus pattern. The amplitude of the bladder pressure evoked at a given location could be modulated by changes in either the amplitude ŽFigs. 1A and 4A,B,C. or the duration ŽFig. 4D. of the stimulus pulse. Greater stimulus intensities produced a relatively uniform shift in the evoked response curves to higher values as well as spread in the dorsoventral extent over which responses were evoked. Similarly, increases in the stimulus frequency or the duration of the stimulus train increased the amplitude of the evoked bladder contractions ŽFig. 2C.. 3.3. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the pericanicular gray Microstimulation in regions around the central canal generated increases in bladder pressure and variable responses in the urethra including decreases in pressure, increases in pressure, and no effect. The bladder and urethral pressures evoked along tracks traversing the pericanicular gray in the S1 and S2 spinal segments Ždifferent animals. are shown in Fig. 3. In both segments pericanicular regions were identified where microstimulation evoked increases in bladder pressure without increases in urethral pressure ŽFig. 3A,B,D.. While the apparent region over which bladder pressures were evoked selectively was larger in the S1 segment ŽFig. 3A,B., the evoked bladder pressures were larger in the S2 segment ŽFig. 3D.. Examples of individual responses evoked at a site where microstimulation evoked strong increases in bladder pressure are illustrated in Fig. 3E and F. The 1 s mapping stimulus evoked an increase in bladder pressure, a weak increase in proximal urethral pressure followed by transmitted pressure from the bladder, and a reduction in pressure in the distal urethra ŽFig. 3E.. Increasing the train duration to 100 s evoked a large, sustained increase in bladder pressure ŽFig. 3F.. Following a short delay, identical pressure responses were observed in both the proximal and distal
Fig. 3. Genitourinary responses to microstimulation of the pericanicular gray in the sacral spinal cord. ŽA, B. Pressures evoked in the bladder, proximal urethra Ž; 6 cm from external meatus., and distal urethra Ž; 3 cm from external meatus. by microstimulation along two penetrations through the S2 segment as shown in panel C. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽC. Tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the locations of the two penetrations ŽA, B.. ŽD. Pressures evoked in the bladder, proximal urethra, and distal urethra by microstimulation along a penetration through the S1 segment, as shown in panel G, in a different experiment. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽE, F. Traces of pressures evoked in the bladder ŽPbl., proximal urethra ŽPup., and distal urethra ŽPud. by microstimulation Žbar. at 2400 mm along track shown in G with either a 1 s ŽE. or 100 s ŽF. 20 Hz train of 100 mA, 100 ms pulses. ŽG. A tracing of the right S1 spinal cord Ž1000 mm per division. illustrating the locations of the penetration and the sites where the individual responses were evoked.
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W.M. Grill et al.r Brain Research 836 (1999) 19–30
urethra. This pattern indicated that the three sensors were equibaric and thus were connected by a fluid column. During this train the animal voided approximately 2 cm3 out of 7 cm3 in the bladder with the urethral catheter in place. These results demonstrate that microstimulation around the central canal produces micturition-like responses in the anesthetized cat. 3.4. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the dorsal aspect of the sacral spinal cord Small and variable bladder pressures were evoked by microstimulation in the dorsal aspect of the spinal cord. This was found both for locations within the dorsal funiculus ŽFig. 4A,B., and in the dorsal horn, particularly the lateral aspect ŽFigs. 1A, 2A and 4C.. Stimulation in the dorsal aspect of the cord, including the dorsal funiculus, also generated increases in urethral pressure. The increases in pressure evoked by dorsal microstimulation were larger at the level of the penis than at the level of the urethral sphincter ŽFigs. 2A and 3A,B.. The urethral pressures evoked by microstimulation in the dorsal aspect of the spinal cord were strongly dependent on the stimulus frequency. At low stimulus frequencies Ž2 Hz. the responses followed 1 for 1 with the stimulus, and had a sustained or slightly decaying amplitude. At intermediate frequencies Ž5–10 Hz. the responses followed 1 for 1 with the stimulus but the amplitude of the response decayed during the stimulus train. The urethral pressures evoked by the 1 s 20 Hz mapping stimulus, and at higher frequencies, were characterized by a strong onset response followed by a rapid decay to a weak or absent sustained response. An example of this type of response is shown in Fig. 4F, and the ubiquity of this response in the dorsal aspect of the cord can be seen in the comparisons in Fig. 4 between the peak and average urethral pressures. This characteristic of the urethral pressure responses was similar to the pressure responses evoked by stimulation of the sacral dorsal roots. In contrast, urethral pressure responses evoked in the ventral aspect of the spinal cord were sustained throughout the stimulus train ŽFig. 4G., and had no frequency dependence beyond what is expected from skeletal muscle w34x. The characteristics of urethral pressures evoked by ventral microstimulation were thus similar in character to pressures evoked by stimulation of the ventral roots.
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4. Discussion A significant component of the neural control of the genitourinary system resides within the sacral spinal cord. In the present experiments the physiological effects within the genitourinary system of electrical activation of spinal neurons were measured. Previous experiments examining the effects of spinal stimulation on the bladder and urethra were limited to stimulation of white matter tracts w15x, to single penetrations directed at the sacral parasympathetic nucleus w9x or pericanicular gray w6x, or employed fixed electrodes at a limited number of locations w24,47x. Similarly, microstimulation to effect voiding in spinal human was limited to single electrodes placed bilaterally in fixed locations w32,33x. The present experiments employed multiple penetrations to identify regions of the sacral spinal cord where microstimulation generated selective contraction of the bladder and regions where microstimulation generated reductions in urethral pressure. The primary limitation of the current results is that the experiments were conducted in a spinal cord intact, anesthetized model. The choice of a-chloralose was based on the considerations that it interferes less with autonomic function as compared to other anesthetics, and largely preserves spinal reflexes w1,7x. Previous data indicate that a-chloralose may effect the magnitude of reflex bladder contractions w39x, as well as synaptic transmission and neural responsiveness within the spinal cord w43x. However, these results provide important baseline maps that can be compared to results from experiments conducted in decerebrate and spinal models. In this preparation, responses could be evoked by direct activation of last-order neurons, as present in the intermediolateral region and ventral horn, and by transsynaptic activation, via stimulation of interneurons, afferent fibers, or descending fibers in the intermediolateral region, pericanicular gray, and dorsal horn. Response could also be evoked by activation of ascending tracts, as in the dorsal funiculus. In the cases of transsynaptic activation it is likely that the chloralose anesthetic reduced the excitability and thus the magnitude of the responses w43x. A second limitation of the current results is that the initial bladder volumes were limited by the presence of distention evoked reflex micturition. Thus, the system was in a quiescent state of low volume, low pressure continence, and the ability to test voiding was limited by the small initial volumes. Further, previous results demonstrate that the effects of afferent inputs,
Fig. 4. Bladder and urethral pressures evoked by intraspinal microstimulation of the S2 segment. ŽA–D. Pressures evoked in the bladder and proximal urethra Ž; 6 cm from external meatus. by microstimulation along four tracks through the S2 spinal segment, as shown in panel E. Stimulus was a 1 s, 20 Hz train of 100 ms at the indicated amplitudes ŽA–C. or 100 mA pulses at the indicated pulse durations ŽD.. Legends in A also apply to B and C. ŽE. A tracing of the right S2 cord Ž1000 mm per division. illustrating the locations of the four penetrations and the sites where the individual responses ŽF, G. were evoked. ŽF, G. Individual urethral pressure responses to microstimulation in the dorsal ŽF. and ventral ŽG. aspects of the spinal cord. Stimuli were 1 s, 20 Hz trains of 100 ms, 100 mA pulses.
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which may have been activated by microstimulation, are highly modulated during the micturition cycle, e.g., Ref. w17x. The largest bladder pressures were generated by microstimulation in the lateral and ventrolateral aspects of the ventral horn. The first region corresponds to the location of the preganglionic parasympathetic axons that innervate the bladder as they course ventrally along the lateral border of the ventral gray from their cell bodies in the intermediolateral cell column w31x. The second region corresponds to where the axons exit the ventral gray to form the a ventral rootlet. These data are similar to those reported by Carter et al. w9x. The bladder pressures evoked by microstimulation in S1 were lower than those evoked by microstimulation in the S2 segment, which is in agreement with anatomical studies demonstrating that the parasympathetic innervation of the bladder is centered in S2 with fewer cells present in S1 w31x. Bladder pressures were also generated by microstimulation along a band just medial to the dorsal root entry zone, and along the lateral border of the dorsal horn. These regions corresponds to areas of dense innervation by somatic afferents in the pudendal nerve w44x and the medial and lateral collateral pathways of pelvic afferents w30x. Pressures evoked in these areas were strongly dependent on stimulus frequency, a characteristic similar to pressure evoked by stimulation of sacral dorsal roots. These data are consistent with the assertion that these pressures were evoked by activation of afferent terminals or second-order relay neurons which generated bladder pressures via direct or indirect synaptic excitation of the preganglionic parasympathetic innervation of the bladder. The magnitude of the bladder pressure increases generated using unilateral microstimulation were comparable to the pressures generated during distension evoked reflex micturition w16,19x or micturition evoked by microstimulation of Barrington’s nucleus w42x. Furthermore, the amplitudes of bladder pressures could be augmented substantially by increases in stimulus frequency or train duration. Examination of the time course of the evoked pressure responses indicated that the increases in bladder pressure were clearly due to contraction of the smooth muscle of the detrusor, rather than the abdominal striated muscle, which would have produced bladder pressures with a rapid onset and offset an order of magnitude faster than those observed here Žsee, e.g., Ref. w27x.. Microstimulation produced both increases and decreases in urethral pressure. The time courses of the changes in urethral pressure indicated that they were mediated predominantly by the activation or relaxation of striated muscle. Furthermore, there is little to no smooth muscle in the region of the proximal urethral sensor w11,46x. Increases in pressure were generated by stimulation within the dorsal horn, in the same regions which generated increases in bladder pressure. The magnitude and characteristics of the urethral pressures evoked by microstimulation were
strongly dependent on stimulus frequency. At lower frequencies the response followed one-for-one with the stimulus and maintained a constant amplitude, while at higher frequencies the amplitude of the response fell sharply after the first or second pulse in the train. These characteristics were similar to urethral pressure responses evoked by stimulation of sacral dorsal roots or pudendal afferents w45x, suggesting that they were evoked indirectly. Electrode locations in the ventral horn produced strong increases in urethral pressure, presumably by direct activation of pudendal motoneurons w44x. Regions where microstimulation generated reductions in urethral pressure included the dorsal aspect of the intermediolateral region and the pericanicular gray. Previous results indicate that these regions contain interneurons which project to pudendal motoneurons w22x, and a recent study also found that stimulation lateral to the central canal generated a reduction in urethral pressure w6x. The mechanism of these reductions in pressure may have been inhibition of ongoing tonic activity or activation of some pelvic floor muscleŽs. which acted to decrease urethral pressure. There are previous results that suggest that the inhibitory mechanism of the external urethral sphincter arises from propriospinal neurons. Electrical stimulation of the surface of the spinal cord Ždorsolateral funiculus. generated inhibition of the urethral sphincter, even after spinal transection w15x, and intracellular recordings from sphincter motoneurons revealed that the latency of descending inhibition is appropriate for an intervening interneuron and not a direct connection w28x. Furthermore, neurons around the central canal in the sacral cord receiving descending input from Barrington’s nucleus contain the inhibitory neurotransmitter GABA w5x, some neurons in the intermediolateral region which are active during reflex micturition are GABAergic w20x, and external urethral sphincter motoneurons are contacted by GABAergic terminals w35,36x. Thus, there exists the neuronal machinery to mediate stimulus evoked inhibition of motoneurons innervating periurethral striated musculature. The stimulus evoked reductions in urethral pressure were only weakly dependent on the stimulus parameters. The limited effect of stimulus parameter on the magnitude of the pressure reductions has two important implications. First, it shows that in this model only limited reductions in urethral pressure could be generated by intraspinal microstimulation with a single microelectrode. Secondly, it demonstrates that the mapping stimulus was adequate to explore regions of the cord which might mediate urethral relaxation, without concern that responses were missed due to the pattern of stimulation. The results of these studies provide a baseline of the physiological responses evoked by intraspinal microstimulation of the sacral spinal cord. Future work should address systematically the influence of anesthetics through use of a decerebrate preparation and the influence of spinalization. Chronic spinalization leads to behavioral w13,37x and ana-
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tomical w3x changes in the sacral spinal neural elements. Therefore, the effects of microstimulation will likely be different in the chronic spinal model.
Acknowledgements This work was supported by the National Institutes of Health, National Institutes on Neurological Disorders and Stroke, Neural Prosthesis Program ŽN01-NS-5-2331, N01NS-8-2300..
References w1x G.U. Balis, R.R. Monroe, The pharmacology of chloralose: a review, Psychopharmacology 6 Ž1964. 1–30. w2x J.G. Banwell, G.H. Creasey, A.M. Aggarwal, D.R. Bodner, J.T. Mortimer, Management of the neurogenic bowel in patients with spinal cord injury, Urol. Clin. North Am. 20 Ž1993. 517–526. w3x M.S. Beattie, M.G. Leedy, J.C. Bresnahan, Evidence for alterations of synaptic inputs to sacral spinal reflex circuits after spinal cord transection in the cat, Exp. Neurol. 123 Ž1. Ž1993. 35–50. w4x B.F.M. Blok, A.T.M. Willemsen, G. Holstege, A PET study on brain control of micturition in humans, Brain 120 Ž1997. 111–121. w5x B.F.M. Blok, H. de Weer, G. Holstege, The pontine micturition center projects to sacral cord GABA immunoreactive neurons in the cat, Neurosci. Lett. 233 Ž1997. 109–112. w6x B.F.M. Blok, J.T. van Maarseveen, G. Holstege, Electrical stimulation of the sacral dorsal gray commissure evokes relaxation of the external urethral sphincter in the cat, Neurosci. Lett. 249 Ž1998. 68–70. w7x G. Bonvento, R. Charbonne, J.-L. Correze, J. Borredon, J. Seylaz, P. Lacombe, In a-chlorolose plus halothane induction a suitable anesthetic regimum for cerebrovascular research?, Brain Res. 665 Ž1994. 213–221. w8x G.S. Brindley, C.E. Polkey, D.N. Rushton, L. Cardozo, Sacral anterior root stimulators for bladder control in paraplegia: the first 50 cases, J. Neurol. Neurosurg. Psychiatry 49 Ž1986. 1104–1114. w9x R.R. Carter, D.B. McCreery, B.J. Woodford, L.A. Bullara, W.F. Agnew, Micturition control by microstimulation of the sacral spinal cord of the cat: acute studies, IEEE Trans. Rehab. Eng. 3 Ž1995. 206–214. w10x G.H. Creasey, D.R. Bodner, Review of electrical stimulation in the management of the neurogenic bladder, Neurorehabilitation 4 Ž1994. 266–274. w11x W.C. Cullen, T.F. Fletcher, W.F. Bradley, Morphometry of the male feline pelvic urethra, J. Urol. 129 Ž1983. 186. w12x W.C. de Groat, A.M. Booth, N. Yoshimura, Neurophysiology of micturition and its modification in animal models of human disease, in: C.A. Maggi ŽEd.., The Autonomic Nervous System, Vol. 3, Harwood Academic Publishers, London, 1993, pp. 227, 290. w13x W.C. de Groat, I. Nadelhaft, R.J. Milne, A.M. Booth, C. Morgan, K. Thor, Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine, J. Auton. Nerv. Syst. 3 Ž1981. 135–160. w14x W.C. de Groat, M.A. Vizzard, I. Araki, J. Roppolo, Spinal interneurons and preganglionic neurons in sacral autonomic reflex pathways, Prog. Brain Res. 107 Ž1996. 97–111. w15x B. Fedirchuk, S.J. Schefchyk, Effects of electrical stimulation of the thoracic spinal cord on bladder and external urethral sphincter activity in the decerebrate cat, Exp. Brain Res. 84 Ž1991. 635–642.
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w16x B. Fedirchuk, S.J. Shefchyk, Membrane potential changes in sphincter motoneurons during micturition in the decerebrate cat, J. Neurosci. 13 Ž1993. 3090–3094. w17x B. Fedirchuk, J.W. Downie, S.J. Shefchyk, Reduction of perineal evoked excitatory postsynaptic potentials in cat lumbar and sacral motoneurons during micturition, J. Neurosci. 14 Ž1994. 6153–6159. w18x W.M. Grill, N. Bhadra, Genitourinary responses to microstimulation of the sacral spinal cord, Soc. Neurosci. Abstr. 22 Ž1996. 1842. w19x W.M. Grill, B. Wang, S. Hadziefendic, M.A. Haxhiu, Identification of the spinal neural network involved in coordination of micturition in the male cat, Brain Res. 796 Ž1998. 150–160. w20x W.M. Grill, S. Hadziefendic, B.O. Erokwu, M.A. Haxhiu, Co-localization of parvalbumin and c-fos in sacral spinal neurons involved in regulation of micturition, Soc. Neurosci. Abstr. 24 Ž1998. 1618. w21x G. Holstege, D. Griffiths, H. De Wall, E. Dalm, Anatomical and physiological observations on supraspinal control of bladder and urethral sphincter muscles in the cat, J. Comp. Neurol. 250 Ž1986. 449–461. w22x G. Holstege, J. Tan, Supraspinal control of motoneurons innervating the striated muscles of the pelvic floor including urethral and anal sphincters in the cat, Brain 110 Ž1987. 1323–1344. w23x C.N. Honda, Visceral and somatic afferent convergence onto neurons near the central canal in the sacral spinal cord of the cat, J. Neurophysiology 53 Ž1985. 1059–1078. w24x U. Jonas, J.P. Heine, E.A. Tanago, Studies on the feasibility of urinary bladder evacuation by direct spinal cord stimulation: I. Parameters of most effective stimulation, Invest. Urol. 13 Ž1975. 142–150. w25x M.N. Kruse, H. Noto, J.R. Roppolo, W.C. de Groat, Pontine control of the urinary bladder and external urethral sphincter in the rat, Brain Res. 532 Ž1990. 182–190. w26x M. Kuru, Nervous control of micturition, Physiol. Rev. 45 Ž1965. 425–494. w27x V.W. Lin, V. Wolfe, F.S. Frost, I. Perkash, Micturition by functional magnetic stimulation, J. Spinal Cord Med. 20 Ž1997. 218–226. w28x R. Mackel, Segmental and descending control of the external urethral and anal sphincters in the cat, J. Physiol. 294 Ž1979. 105–122. w29x S.B. McMahon, J.F.B. Morrison, Two groups of spinal interneurons that respond to stimulation of the abdominal viscera of the cat, J. Physiol. 322 Ž1982. 1–20. w30x C. Morgan, I. Nadelhaft, W.C. de Groat, The distribution of visceral primary afferents from the pelvic nerve to Lissauer’s tract and the spinal grey matter and its relationship to the sacral parasympathetic nucleus, J. Comp. Neurol. 201 Ž1981. 415–440. w31x I. Nadelhaft, W.C. DeGroat, C. Morgan, Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of HRP, J. Comp. Neurol. 193 Ž1980. 265–286. w32x B.S. Nashold, H. Friedman, S. Boyarsky, Electrical activation of micturition by spinal cord stimulation, J. Surg. Res. 11 Ž1971. 144–147. w33x B.S. Nashold, H. Friedman, J. Grimes, Electrical stimulation of the conus medullaris to control the bladder in the paraplegic patient, Appl. Neurophys. 44 Ž1981. 225–232. w34x P.M.H. Rack, D.R. Westbury, The effects of length and stimulus rate on tension in the isometric cat soleus muscle, J. Physiol. 204 Ž1969. 443–460. w35x V. Ramirez-Leon, B. Ulfhake, GABA-like immunoreactive innervation and dendro-dendritic contacts in the ventrolateral dendritic bundle in the cat S1 spinal cord segment: an electron microscopic study, Exp. Brain Res. 97 Ž1993. 1–12. w36x V. Ramirez-Leon, B. Ulfhake, U. Arvidsson, A.A. Verhofstad, T.J. Visser, T. Hokfelt, Serotoninergic, peptidergic and GABAergic innervation of the ventrolateral and dorsolateral motor nuclei in the cat S1rS2 segments: an immunofluorescence study, J. Chem. Neuroanat. 7 Ž1994. 87–103. w37x G. Rampal, P. Mignard, Behavior of the urethral striated sphincter
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Research report
Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats Warren M. Grill ) , Narendra Bhadra, Baoqing Wang Department of Biomedical Engineering, Applied Neural Control Laboratory, C.B. Bolton Building, Room 3480, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106-4912, USA Accepted 4 May 1999
Abstract Experiments were conducted to measure the bladder and urethral pressures evoked by intraspinal microstimulation of the sacral segments ŽS1–S2. in neurologically intact, chloralose anesthetized adult male cats. The bladder pressure was measured with a superpubic catheter and the urethral pressure was measured simultaneously at the level of the urethral sphincter and at the level of the penis using a two-element micromanometer. Intraspinal stimuli Žtypically 1 s, 20 Hz, 100 mA, 100 ms. were applied with activated iridium microwire electrodes in ipsilateral segments and intersegmental boundaries with a 250 mm mediolateral resolution and a 200 mm dorsoventral resolution. Increases in bladder pressures were generated by microstimulation in the intermediolateral region, in the lateral and ventrolateral ventral horn, and around the central canal. Simultaneous increases in urethral pressure were evoked by microstimulation in the ventrolateral ventral horn, but not at the other locations. Small reductions in urethral pressure Ž- 10 cm H 2 O. were evoked at locations in the intermediate laminae and around the central canal. The magnitude of these pressure reductions was weakly dependent on the stimulus parameters. Stimulation around the central canal produced bladder contractions with either no change or a reduction in urethral pressure and voiding of small amounts of fluid. These results demonstrate that regions are present in the spinal intact anesthetized cat where microstimulation generates selective contraction of the bladder without increases in urethral pressure and that regions are present where microstimulation generates small reductions in urethral pressure. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micturition; Genitourinary; Parasympathetic; Electrical stimulation; Neural prosthesis
1. Introduction The genitourinary system is a complex of smooth and striated muscles controlled by somatic and autonomic innervation, including both parasympathetic and sympathetic components w12x. Control of storage and release of urine, the primary functions of the genitourinary system, includes supraspinal, spinal, and ganglionic elements. It has been demonstrated in animals that the normal cycle of micturition involves a spinal–pontine–spinal reflex loop in which descending commands orchestrate contraction of the smooth muscle detrusor and relaxation of periurethral striated musculature w21,25x. Lesion studies w26,40x and a recent imaging study w4x support a role for the dorsolateral pons in control of micturition in the human. ) Corresponding [email protected]
author.
Fax:
q 1-216-368-4872;
E-mail:
Spinal cord injury disrupts these reflex loops and leads to changes in the functioning of the system, including an inability to void, hyperreflexia, and bladder–sphincter dyssynergia, which can lead to serious secondary complications including infection, upper urinary tract damage, and hydronephrosis w2x. Electrical activation of intact neuronal structures below the level of the lesion has been investigated for restoration of micturition in persons with spinal cord injury w10,38x. Initial efforts to electrically evacuate the bladder were largely unsuccessful due to concomitant activation of the periurethral striated musculature both by the electrical stimulus and by local reflex actions w41x. A number of methods including pudendal neurectomy, sphincterotomy, and direct bladder stimulation have been attempted to avoid sphincter activation. The most successful to date has been intermittent stimulation of the sacral ventral roots, in which urine flows during the inter-burst interval, combined with sacral dorsal rhizotomy
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 5 8 1 - 4
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w8x. Another method that met with mixed success was direct activation of the ‘‘micturition center’’ in the spinal cord using penetrating microelectrodes w32,33x. Although anatomical and physiological studies in animals have not demonstrated the existence of a spinal micturition center, it may be possible to activate spinal neurons to produce physiological voiding in individuals with spinal cord injury. Importantly, the early human studies demonstrated the feasibility of chronic intraspinal microstimulation. The possibility of restoring bladder function through intraspinal microstimulation holds promise and has not been systematically investigated. Furthermore, the physiological roles of different groups of spinal neurons in control of micturition have yet to be completely understood w13,14,23,29x. The objective of the experiments reported here was to measure the pressures produced in the genitourinary system by microstimulation of the sacral spinal cord. The goal was to determine whether regions could be identified where microstimulation generated selective contraction of the bladder, and whether regions could be identified where microstimulation generated reductions in urethral pressure. Experiments were conducted in spinal-intact animals and these results serve as a baseline against which responses generated by microstimulation in chronic spinal model can be evaluated. Preliminary results have been reported in abstract form w18x.
2. Materials and methods 2.1. Experimental preparation Acute experiments were conducted on adult, sexually intact, male cats Ž2.8–4.5 kg.. All animal care and experimental procedures were according to NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. Animals were sedated with xylazine ŽRompun, 2.0 mgrkg, s.q.. and anesthetized with ketamine HCl ŽKetaset, 15 mgrkg, i.m., supplemented as required during surgical preparation.. A venous catheter was inserted in the cephalic vein, and anesthesia maintained with a-chloralose ŽSigma, 60 mgr kg, i.v., supplemented at 15 mgrkg.. A midline abdominal incision was made to expose the bladder, the ureters were ligated, transected proximal to the ligation, and drained externally. The bladder was cannulated with a PE tube introduced with a hypodermic needle and secured with a purse string suture. A vent tube, open to atmosphere, was placed adjacent to the bladder, and the abdominal incision was closed in layers. A laminectomy was made to expose the sacral spinal cord and sacral spinal roots. The animal was mounted in a frame with pins at the hips, the head in a headholder, and a vertebral clamp at L4. Body temperature was maintained between 378 and 398C with a thermostatically controlled heat lamp, warm 0.9% saline with 8.4 mgrcm3 sodium bicarbonate and 5% dextrose added was
administered i.v. Ž; 20 cm3rh., respiration was maintained with a respirator, and the electrocardiogram was monitored throughout the experiment. Dexamethasone Ž2 mgrkg, i.v.. was administered at the completion of the laminectomy and every 6 h thereafter for the duration of the experiment. 2.2. Recording and stimulation Bladder pressure was measured using a solid state pressure transducer connected to the superpubic PE catheter ŽDeltran DPT-100, Utah Medical Products, Midvale, UT.. Bladder volume was maintained at a level Ž5–10 cm3 . that was below the threshold to generate reflex contractions of the bladder, and thus all measurements were made during a low volume, low pressure continence phase of the micturition cycle. Urethral pressures were measured using a custom two-channel micromanometer which had two directionally oriented solid state pressure transducers, spaced 3 cm apart and circumferentially offset by 1808, mounted in a silicone rubber catheter 1.4 mm in diameter ŽModel CTrS2, MMI, Hackensack, NJ.. The catheter was used to measure simultaneously the pressure in the sphincteric and penile portions of the urethra. The catheter was positioned by slowly withdrawing it along the length of the urethra to find the location of maximum pressure. The pressure at this location also exhibited spontaneous fluctuations indicative of the activity of the external urethral sphincter and lay approximately 6 cm from the external meatus w46x. The second sensor was displaced 3 cm distally and lay within the penile urethra. In two experiments, urethral pressures were measured with a fluid-filled PE tubing catheter Žo.d.s 0.96 mm., with a sealed tip and a sidehole of diameter ; 0.8 mm, connected to a solid state pressure transducer. The pressure signals were amplified, low pass filtered Žcut-off frequencys 30 Hz., and continuously recorded on a strip chart recorder. Pressures evoked by stimulation were also sampled by computer Žsampling frequencys 100 Hz.. The dura was opened and the individual dorsal and ventral sacral roots were isolated and stimulated using a hook electrode and constant current stimulator Ž1 s, 20 Hz, 100 ms, 1 mA.. The root that produced the largest bladder pressure was presumed to be S2. This identification was tested by stimulation of the more rostral root, which generated much larger urethral pressures, and confirmed by post-mortem dissection to identify the root exits from the spinal column. After root identification, the cord was covered with warm mineral oil and mapping by microstimulation commenced. Vertical, dorsal-to-ventral penetrations Ždepth increments 100–400 mm, typically 200 mm. were made at multiple mediolateral locations between the midline and the lateral border Ždistance between parallel tracks was typically 250 mm. in the rostrocaudal middle of the S1 Ž n s 17 penetrations across two experiments. and S2 Ž n s 55 penetrations across eight experiments. seg-
W.M. Grill et al.r Brain Research 836 (1999) 19–30
ments, and at intersegmental boundaries between S1 and S2 Ž n s 16 penetrations across two experiments. and S2 and S3 Ž n s 6 penetrations in one experiment.. Activated iridium wire microelectrodes Ž50 mm diameter. with an exposed electrochemically determined surface area of ; 225 mm2 , a 1–3 mm tip, and insulated with Epoxylite were
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used for most penetrations ŽIS300, Huntington Medical Research Institutes, Pasadena, CA., although some 75 mm parylene-insulated iridium wire electrodes with an exposed geometric surface area of 250 mm2 and a 1–2 mm tip were also used ŽIR3PT100, MicroProbe, Clarksburg, MD.. Stimuli were charge balanced biphasic pulses with a ca-
Fig. 1. Bladder and urethral pressures evoked by intraspinal microstimulation of the rostral S2 intermediolateral region and lateral ventral horn. ŽA. Pressures evoked in the bladder and proximal urethra Ž; 6 cm from the external meatus. by intraspinal microstimulation with a 1 s 20 Hz train of 100 ms pulses at the indicated amplitudes. The ‘‘peak of slow component’’ points indicate urethral pressure increases attributable to transmitted pressures from the bladder. ŽB, C. Individual bladder and urethral pressure responses evoked by microstimulation at depths of 2400 mm ŽB. or 3400 mm ŽC. with a 1 s, 20 Hz train of 100 ms, 200 mA pulses. ŽD. Tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the location of the penetration and the locations where the individual responses were evoked ŽB, C..
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thodic phase amplitude of 10–200 mA and duration of 50–400 ms applied as 1 s to 10 s continuous and intermittent trains with a frequency between 2 Hz and 100 Hz. The amplitude of the anodic phase was limited to 100 mA and the duration was set automatically by the stimulator to balance the charge injected in the primary phase. The
standard mapping stimulus was a 1 s train of 100 mA, 100 ms, 20 Hz pulses. At the conclusion of the experiment the spinal cord was fixed in situ by immersion in 10% buffered formalin and the animal was killed by an overdose of sodium pentobarbital. The spinal segments of interest were excised and
Fig. 2. Bladder and urethral pressures evoked by intraspinal microstimulation of the caudal S2 intermediolateral region and lateral ventral horn. ŽA. Pressures evoked in the bladder, proximal urethra, and distal urethra by microstimulation along a penetration through the S2 intermediolateral region. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽB. Traces of the pressures evoked in the bladder ŽPbl., proximal urethra ŽPup, ; 6 cm from external meatus., and distal urethra ŽPud, ; 3 cm from external meatus. by microstimulation at 2200 mm Ža. and 2400 mm Žb. below the surface. Stimulus was a 1 s 20 Hz train of 100 ms, 100 mA pulses. ŽC. Influence of stimulus parameters on the pressures evoked in the bladder ŽPbl., proximal urethra ŽPup., and distal urethra ŽPud. by microstimulation in the S2 spinal cord. The stimulus was located at 1500 mm microns below the surface in the dorsal aspect of the intermediolateral region. ŽD. A tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the location of the penetration and the locations where the individual responses were evoked ŽBa, Bb, C..
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placed in refrigerated 10% buffered formalin. At the completion of mapping a particular transverse segment in some experiments, the electrode was moved contralateral to the region mapped, inserted to a known depth, and cut-off. These electrodes remained in the cord throughout fixation and blocking to identify the rostrocaudal location of the penetrations. Spinal cord sections were blocked, microscopic images were digitized, and outlines of the white matter and gray matter were made using a computer-based image analysis system. Bladder pressures were measured as the peak of the evoked pressure with respect to the baseline pressure in the bladder, the latter averaged over the 1 s interval preceding the stimulus. Urethral pressures were measured as the mean of the pressure evoked during the stimulus train with respect to the baseline pressure in the urethra averaged over the 1 s interval preceding the stimulus. Thus, all pressures are reported as changes relative to the pre-stimulus baseline value, rather than as absolute values. The baseline pressures in the bladder varied from 1 to 21 cm H 2 O and exhibited little variation within an experiment Žaverage range s 5 cm H 2 O.. The baseline pressures in the proximal urethra varied from 9 to 30 cm H 2 O and had greater variation during the experiment Žaverage range s 13 cm H 2 O. than the baseline bladder pressure.
3. Results Genitourinary responses to microstimulation of the sacral spinal cord were assessed by measuring pressure changes in the bladder, proximal Žsphincteric. urethra, and distal Žpenile. urethra. Most penetrations were made in the region of the intermediolateral cell column of the S2 segment, and results are presented based on 94 penetration across eight experiments in which the average number of penetrations in the sacral segments was 13 and the range was 3–22. In some experiments, the hindlimb motor responses evoked by intraspinal stimulation in the lumbar segments were also measured, but these data will be reported separately. The 1-s 20-Hz mapping stimulus produced a monophasic increase in bladder pressure with a time to peak of 2.0–2.5 s, followed by a slow decay to baseline ŽFigs. 1B,C and 2B,C.. Bladder pressures were quantified by measuring the peak amplitude of this pressure increase relative to the baseline pressure preceding the stimulus. Increases ŽFigs. 1C and 2B. or decreases ŽFig. 2C. in urethral pressure were generated with short latency in both the region of the sphincter and in the penis. Urethral pressures were quantified by measuring the average pressure during the stimulus train, relative to the baseline pressure preceding the stimulus. As little striated muscle is present along the penile urethra w46x, the rapid pressure changes in the penile urethra were presumed to be due to activation of the ischiocavernosus and bulbocavernosus
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muscles at the base of the penis. This presumption was supported by the observation that penile pressure increases were correlated with twitching of the penis. 3.1. Segmental distribution of pressure responses Bladder and urethral pressures were evoked by microstimulation at locations spread throughout the sacral segments. The magnitude and characteristics of the pressure responses varied according to the location ŽTable 1., magnitude, and pattern of the stimulus. Larger bladder pressures were evoked by microstimulation within the S2 segment than by microstimulation within the S1 segment, although the difference was not significant. This was consistent with the higher bladder pressures evoked by stimulation of the S2 ventral than the S1 ventral root Ž p 0.0001, t-test of the null hypothesis that the means pressures were the same from S1 and S2 stimulation.. Conversely, larger urethral pressures at the level of the urethral sphincter were evoked by microstimulation within the S1 segment than within the S2 segment, although the difference was not significant. This also was consistent with the higher urethral pressures evoked by stimulation of the S1 ventral root than the S2 ventral root, although this difference was also not significant Ž p s 0.0861, t-test of the null hypothesis that the means pressures were the same from S1 and S2 stimulation.. Thus, the magnitudes of the bladder and urethral pressures evoked by intraspinal microstimulation differed across spinal segments and were correlated with the pressures evoked by spinal root stimulation. 3.2. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the intermediolateral region The amplitude and characteristics of the pressure responses evoked by microstimulation were dependent on the dorsoventral as well as the mediolateral location of the stimulus. An example of an electrode penetration traversing the intermediolateral region and lateral ventral horn of the S2 segment is shown in Fig. 1. Bladder pressures greater than 15 cm H 2 O were consistently evoked by microstimulation in the intermediolateral region and in the
Table 1 Bladder and urethral pressures evoked by intraspinal microstimulation and spinal root stimulation Entries are mean"S.D. Žcm H 2 O. Ž n.. Bladder a
Intraspinal Dorsal root b Ventral root b a b
Urethra
S1
S2
S1
S2
21"17 Ž3. 8"7 Ž6. 9"7 Ž7.
28"9 Ž8. 22"21 Ž6. 43"11 Ž7.
94"73 Ž3. 40"46 Ž6. 66"52 Ž7.
75"77 Ž8. 13"10 Ž6. 20"29 Ž7.
Maximum pressures evoked by 1 s, 20 Hz, 100 mA, 100 ms stimulus. Maximum pressures evoked by 1 s, 20 Hz, 1 mA, 100 ms stimulus.
24
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W.M. Grill et al.r Brain Research 836 (1999) 19–30
lateral and ventrolateral aspects of the ventral horn. No activation of periurethral striated muscle was evoked by microstimulation in the intermediolateral region or the lateral ventral horn. Microstimulation in the intermediolateral region evoked a slow increase in urethral pressure at the level of the urethral sphincter following stimulation ŽFig. 1B.. This slow, long-latency response was the result of transmitted pressure from the bladder via fluid flow from the bladder to the urethra. Generation of large bladder pressures without increases in urethral pressure by microstimulation in the intermediolateral region and the lateral ventral horn can also be seen in Fig. 4C and D, respectively, as well as in Fig. 2. Microstimulation at locations in the ventrolateral aspect of the ventral horn generated large bladder pressures, but also generated increases in urethral pressure. As shown in Fig. 1C, there was a short-latency increase in urethral pressure which subsided on termination of the stimulus, followed by a slow, long-latency pressure increase transmitted from the bladder. A similar distinction is illustrated in Fig. 2. In this case the baseline pressure proximal to the sensor was sufficient to prevent transmitted pressure from being measured. However, there was a clear distinction between areas in the lateral ventral horn that generated selective activation of the bladder ŽFig. 2Ba., and areas in the ventral aspect of the ventral horn that generated coactivation of the bladder and periurethral striated muscle ŽFig. 2Bb.. These data demonstrate that microstimulation in the area of the intermediolateral region and the lateral ventral horn generated large bladder pressures Ž30–40 cm H 2 O., without co-activation of periurethral musculature. Microstimulation in the ventral aspect of the ventral horn generated large increases in bladder pressure accompanied by large increases in urethral pressure, both at the level of the urethral sphincter and in the penile urethra. Microstimulation in the dorsal aspect of the intermediolateral region produced increases in bladder pressure coupled with small reductions in urethral pressure ŽFig. 2.. Stimulus locations that generated a reduction in urethral pressure were of particular interest, as a drop in urethral pressure accompanies natural micturition. The mapping stimulus generated reductions in pressure of less than 10 cm H 2 O and the influence of stimulus parameters was explored in an attempt to augment these pressure reductions ŽFig. 2C.. In this example, the mapping stimulus generated a small reduction in pressure the proximal urethra, and no change in pressure in the distal urethra.
25
Increasing the stimulus amplitude resulted in an increase in the evoked bladder pressure, but did not augment the reduction in urethral pressure. The magnitude of the pressure reduction increased as the stimulus frequency was increased, but reached a plateau at frequencies greater than 40 Hz. Applying longer trains at either 100% or 50% duty cycle did not further augment the pressure reduction. These results show that the stimulus evoked reductions in urethral pressure were only weakly dependent on the stimulus pattern. The amplitude of the bladder pressure evoked at a given location could be modulated by changes in either the amplitude ŽFigs. 1A and 4A,B,C. or the duration ŽFig. 4D. of the stimulus pulse. Greater stimulus intensities produced a relatively uniform shift in the evoked response curves to higher values as well as spread in the dorsoventral extent over which responses were evoked. Similarly, increases in the stimulus frequency or the duration of the stimulus train increased the amplitude of the evoked bladder contractions ŽFig. 2C.. 3.3. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the pericanicular gray Microstimulation in regions around the central canal generated increases in bladder pressure and variable responses in the urethra including decreases in pressure, increases in pressure, and no effect. The bladder and urethral pressures evoked along tracks traversing the pericanicular gray in the S1 and S2 spinal segments Ždifferent animals. are shown in Fig. 3. In both segments pericanicular regions were identified where microstimulation evoked increases in bladder pressure without increases in urethral pressure ŽFig. 3A,B,D.. While the apparent region over which bladder pressures were evoked selectively was larger in the S1 segment ŽFig. 3A,B., the evoked bladder pressures were larger in the S2 segment ŽFig. 3D.. Examples of individual responses evoked at a site where microstimulation evoked strong increases in bladder pressure are illustrated in Fig. 3E and F. The 1 s mapping stimulus evoked an increase in bladder pressure, a weak increase in proximal urethral pressure followed by transmitted pressure from the bladder, and a reduction in pressure in the distal urethra ŽFig. 3E.. Increasing the train duration to 100 s evoked a large, sustained increase in bladder pressure ŽFig. 3F.. Following a short delay, identical pressure responses were observed in both the proximal and distal
Fig. 3. Genitourinary responses to microstimulation of the pericanicular gray in the sacral spinal cord. ŽA, B. Pressures evoked in the bladder, proximal urethra Ž; 6 cm from external meatus., and distal urethra Ž; 3 cm from external meatus. by microstimulation along two penetrations through the S2 segment as shown in panel C. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽC. Tracing of the right S2 spinal cord Ž1000 mm per division. illustrating the locations of the two penetrations ŽA, B.. ŽD. Pressures evoked in the bladder, proximal urethra, and distal urethra by microstimulation along a penetration through the S1 segment, as shown in panel G, in a different experiment. Stimulus was a 1 s, 20 Hz train of 100 ms, 100 mA pulses. ŽE, F. Traces of pressures evoked in the bladder ŽPbl., proximal urethra ŽPup., and distal urethra ŽPud. by microstimulation Žbar. at 2400 mm along track shown in G with either a 1 s ŽE. or 100 s ŽF. 20 Hz train of 100 mA, 100 ms pulses. ŽG. A tracing of the right S1 spinal cord Ž1000 mm per division. illustrating the locations of the penetration and the sites where the individual responses were evoked.
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W.M. Grill et al.r Brain Research 836 (1999) 19–30
urethra. This pattern indicated that the three sensors were equibaric and thus were connected by a fluid column. During this train the animal voided approximately 2 cm3 out of 7 cm3 in the bladder with the urethral catheter in place. These results demonstrate that microstimulation around the central canal produces micturition-like responses in the anesthetized cat. 3.4. Bladder and urethral pressures eÕoked by intraspinal microstimulation in the dorsal aspect of the sacral spinal cord Small and variable bladder pressures were evoked by microstimulation in the dorsal aspect of the spinal cord. This was found both for locations within the dorsal funiculus ŽFig. 4A,B., and in the dorsal horn, particularly the lateral aspect ŽFigs. 1A, 2A and 4C.. Stimulation in the dorsal aspect of the cord, including the dorsal funiculus, also generated increases in urethral pressure. The increases in pressure evoked by dorsal microstimulation were larger at the level of the penis than at the level of the urethral sphincter ŽFigs. 2A and 3A,B.. The urethral pressures evoked by microstimulation in the dorsal aspect of the spinal cord were strongly dependent on the stimulus frequency. At low stimulus frequencies Ž2 Hz. the responses followed 1 for 1 with the stimulus, and had a sustained or slightly decaying amplitude. At intermediate frequencies Ž5–10 Hz. the responses followed 1 for 1 with the stimulus but the amplitude of the response decayed during the stimulus train. The urethral pressures evoked by the 1 s 20 Hz mapping stimulus, and at higher frequencies, were characterized by a strong onset response followed by a rapid decay to a weak or absent sustained response. An example of this type of response is shown in Fig. 4F, and the ubiquity of this response in the dorsal aspect of the cord can be seen in the comparisons in Fig. 4 between the peak and average urethral pressures. This characteristic of the urethral pressure responses was similar to the pressure responses evoked by stimulation of the sacral dorsal roots. In contrast, urethral pressure responses evoked in the ventral aspect of the spinal cord were sustained throughout the stimulus train ŽFig. 4G., and had no frequency dependence beyond what is expected from skeletal muscle w34x. The characteristics of urethral pressures evoked by ventral microstimulation were thus similar in character to pressures evoked by stimulation of the ventral roots.
27
4. Discussion A significant component of the neural control of the genitourinary system resides within the sacral spinal cord. In the present experiments the physiological effects within the genitourinary system of electrical activation of spinal neurons were measured. Previous experiments examining the effects of spinal stimulation on the bladder and urethra were limited to stimulation of white matter tracts w15x, to single penetrations directed at the sacral parasympathetic nucleus w9x or pericanicular gray w6x, or employed fixed electrodes at a limited number of locations w24,47x. Similarly, microstimulation to effect voiding in spinal human was limited to single electrodes placed bilaterally in fixed locations w32,33x. The present experiments employed multiple penetrations to identify regions of the sacral spinal cord where microstimulation generated selective contraction of the bladder and regions where microstimulation generated reductions in urethral pressure. The primary limitation of the current results is that the experiments were conducted in a spinal cord intact, anesthetized model. The choice of a-chloralose was based on the considerations that it interferes less with autonomic function as compared to other anesthetics, and largely preserves spinal reflexes w1,7x. Previous data indicate that a-chloralose may effect the magnitude of reflex bladder contractions w39x, as well as synaptic transmission and neural responsiveness within the spinal cord w43x. However, these results provide important baseline maps that can be compared to results from experiments conducted in decerebrate and spinal models. In this preparation, responses could be evoked by direct activation of last-order neurons, as present in the intermediolateral region and ventral horn, and by transsynaptic activation, via stimulation of interneurons, afferent fibers, or descending fibers in the intermediolateral region, pericanicular gray, and dorsal horn. Response could also be evoked by activation of ascending tracts, as in the dorsal funiculus. In the cases of transsynaptic activation it is likely that the chloralose anesthetic reduced the excitability and thus the magnitude of the responses w43x. A second limitation of the current results is that the initial bladder volumes were limited by the presence of distention evoked reflex micturition. Thus, the system was in a quiescent state of low volume, low pressure continence, and the ability to test voiding was limited by the small initial volumes. Further, previous results demonstrate that the effects of afferent inputs,
Fig. 4. Bladder and urethral pressures evoked by intraspinal microstimulation of the S2 segment. ŽA–D. Pressures evoked in the bladder and proximal urethra Ž; 6 cm from external meatus. by microstimulation along four tracks through the S2 spinal segment, as shown in panel E. Stimulus was a 1 s, 20 Hz train of 100 ms at the indicated amplitudes ŽA–C. or 100 mA pulses at the indicated pulse durations ŽD.. Legends in A also apply to B and C. ŽE. A tracing of the right S2 cord Ž1000 mm per division. illustrating the locations of the four penetrations and the sites where the individual responses ŽF, G. were evoked. ŽF, G. Individual urethral pressure responses to microstimulation in the dorsal ŽF. and ventral ŽG. aspects of the spinal cord. Stimuli were 1 s, 20 Hz trains of 100 ms, 100 mA pulses.
28
W.M. Grill et al.r Brain Research 836 (1999) 19–30
which may have been activated by microstimulation, are highly modulated during the micturition cycle, e.g., Ref. w17x. The largest bladder pressures were generated by microstimulation in the lateral and ventrolateral aspects of the ventral horn. The first region corresponds to the location of the preganglionic parasympathetic axons that innervate the bladder as they course ventrally along the lateral border of the ventral gray from their cell bodies in the intermediolateral cell column w31x. The second region corresponds to where the axons exit the ventral gray to form the a ventral rootlet. These data are similar to those reported by Carter et al. w9x. The bladder pressures evoked by microstimulation in S1 were lower than those evoked by microstimulation in the S2 segment, which is in agreement with anatomical studies demonstrating that the parasympathetic innervation of the bladder is centered in S2 with fewer cells present in S1 w31x. Bladder pressures were also generated by microstimulation along a band just medial to the dorsal root entry zone, and along the lateral border of the dorsal horn. These regions corresponds to areas of dense innervation by somatic afferents in the pudendal nerve w44x and the medial and lateral collateral pathways of pelvic afferents w30x. Pressures evoked in these areas were strongly dependent on stimulus frequency, a characteristic similar to pressure evoked by stimulation of sacral dorsal roots. These data are consistent with the assertion that these pressures were evoked by activation of afferent terminals or second-order relay neurons which generated bladder pressures via direct or indirect synaptic excitation of the preganglionic parasympathetic innervation of the bladder. The magnitude of the bladder pressure increases generated using unilateral microstimulation were comparable to the pressures generated during distension evoked reflex micturition w16,19x or micturition evoked by microstimulation of Barrington’s nucleus w42x. Furthermore, the amplitudes of bladder pressures could be augmented substantially by increases in stimulus frequency or train duration. Examination of the time course of the evoked pressure responses indicated that the increases in bladder pressure were clearly due to contraction of the smooth muscle of the detrusor, rather than the abdominal striated muscle, which would have produced bladder pressures with a rapid onset and offset an order of magnitude faster than those observed here Žsee, e.g., Ref. w27x.. Microstimulation produced both increases and decreases in urethral pressure. The time courses of the changes in urethral pressure indicated that they were mediated predominantly by the activation or relaxation of striated muscle. Furthermore, there is little to no smooth muscle in the region of the proximal urethral sensor w11,46x. Increases in pressure were generated by stimulation within the dorsal horn, in the same regions which generated increases in bladder pressure. The magnitude and characteristics of the urethral pressures evoked by microstimulation were
strongly dependent on stimulus frequency. At lower frequencies the response followed one-for-one with the stimulus and maintained a constant amplitude, while at higher frequencies the amplitude of the response fell sharply after the first or second pulse in the train. These characteristics were similar to urethral pressure responses evoked by stimulation of sacral dorsal roots or pudendal afferents w45x, suggesting that they were evoked indirectly. Electrode locations in the ventral horn produced strong increases in urethral pressure, presumably by direct activation of pudendal motoneurons w44x. Regions where microstimulation generated reductions in urethral pressure included the dorsal aspect of the intermediolateral region and the pericanicular gray. Previous results indicate that these regions contain interneurons which project to pudendal motoneurons w22x, and a recent study also found that stimulation lateral to the central canal generated a reduction in urethral pressure w6x. The mechanism of these reductions in pressure may have been inhibition of ongoing tonic activity or activation of some pelvic floor muscleŽs. which acted to decrease urethral pressure. There are previous results that suggest that the inhibitory mechanism of the external urethral sphincter arises from propriospinal neurons. Electrical stimulation of the surface of the spinal cord Ždorsolateral funiculus. generated inhibition of the urethral sphincter, even after spinal transection w15x, and intracellular recordings from sphincter motoneurons revealed that the latency of descending inhibition is appropriate for an intervening interneuron and not a direct connection w28x. Furthermore, neurons around the central canal in the sacral cord receiving descending input from Barrington’s nucleus contain the inhibitory neurotransmitter GABA w5x, some neurons in the intermediolateral region which are active during reflex micturition are GABAergic w20x, and external urethral sphincter motoneurons are contacted by GABAergic terminals w35,36x. Thus, there exists the neuronal machinery to mediate stimulus evoked inhibition of motoneurons innervating periurethral striated musculature. The stimulus evoked reductions in urethral pressure were only weakly dependent on the stimulus parameters. The limited effect of stimulus parameter on the magnitude of the pressure reductions has two important implications. First, it shows that in this model only limited reductions in urethral pressure could be generated by intraspinal microstimulation with a single microelectrode. Secondly, it demonstrates that the mapping stimulus was adequate to explore regions of the cord which might mediate urethral relaxation, without concern that responses were missed due to the pattern of stimulation. The results of these studies provide a baseline of the physiological responses evoked by intraspinal microstimulation of the sacral spinal cord. Future work should address systematically the influence of anesthetics through use of a decerebrate preparation and the influence of spinalization. Chronic spinalization leads to behavioral w13,37x and ana-
W.M. Grill et al.r Brain Research 836 (1999) 19–30
tomical w3x changes in the sacral spinal neural elements. Therefore, the effects of microstimulation will likely be different in the chronic spinal model.
Acknowledgements This work was supported by the National Institutes of Health, National Institutes on Neurological Disorders and Stroke, Neural Prosthesis Program ŽN01-NS-5-2331, N01NS-8-2300..
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