- Email: [email protected]
Urinary Bladder Reinnervation
0022-534 7/86/1364-0964$02.00/() Vol. 136, October Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1986 by The Williams & Wilkins Co.
URINARY BLADDER REINNERVATION BERT VORSTMAN,* STEVEN M. SCHLOSSBERG, LEONARD KASS
AND
CHARLES J. DEVINE, JR.
From the Departments of Urology and Physiology, Eastern Virginia Medical School, Norfolk, Virginia
ABSTRACT
The ability of mixed spinal nerve roots to regenerate and reinnervate the urinary bladder was examined in young adult female cats. Using microsurgical technique, a unilateral extradural spinal nerve root anastomosis of a lumbar (L 7) to a sacral root (Sl) either with or without a nerve graft was performed. Remaining ipsilateral sacral roots were transected. The contralateral normal sacral roots remained intact and allowed the animals adequate urination during the period necessary for axonal regeneration. At the time of restudy seven months later, stimulation of the anastomosed nerve root proximal to the anastomosis (isolated from the spinal cord) elicited a bladder contraction. Significant lumbar axonal regeneration was substantiated by compound action potentials recorded across the anastomosis. In addition, redirection of axons from a lumbar to a sacral distribution was demonstrated. The contralateral normal sacral roots provided control cystometric and electrophysiological data against which responses from the previously anastomosed nerve roots were compared. In conclusion, significant bladder reinnervation can occur after an anastomosis of a lumbar and sacral root with or without a nerve graft. This technique, or variations thereof, may have a clinical role in selected patients with neurogenic bladder dysfunction to reinnervate the bladder and restore central control. Current treatment of the decentralized bladder has decreased the morbidity and mortality associated with the condition but has not realized functional micturition. 1 In fact, research into restoring central control of the neurogenic bladder has been limited. Attempts at urinary bladder reinnervation have been described and, although first conceptualized in 1907, few studies have examined cystometric and compound action potential data to analyze systematically urinary bladder reinnervation. 2 Both intradural and extradural nerve root crossover surgical techniques have been performed in animal models and a few studies on paraplegic and spina bifida patients have also been reported.2-5 Recently, an experimental study was described that documented the return of the micturition reflex after an intradural microsurgical reconstruction of sacral motor and sensory roots. 6 Urinary bladder reinnervation or recentralization through the use of mixed spinal nerve roots and nerve grafts has not been reported previously. Nerve grafts would likely be necessary for bypassing an injured segment of the spinal cord for reinnervation procedures in humans because of the distance to be bridged between functional nerve roots above the injured segment and sacral roots below. In the present work, the following questions were investigated: a) whether electrophysiologic and cystometric data would indicate axonal regeneration and bladder reinnervation after a mixed suprasacral nerve root (L 7) to mixed sacral root (81) anastomosis; b) whether similar data might be obtained after a nerve root anastomosis via a nerve graft; and c) whether a lumbar to sacral mixed root anastomosis would provide for redirection of axons. MATERIALS AND METHODS
Initial surgery. Eleven adult female cats (two to three kg.) were anesthetized with intramuscular ketamine (25 mg./kg.). An intravenous line was inserted for administration of normal saline, ampicillin and incremental doses of phenobarbital. The animals were intubated and connected to a volume ventilator Accepted for publication May 9, 1986. * Requests for reprints: Department of Urology, University of Miami School of Medicine, Miami, FL 33101. Supported by American Paralysis Association grant BWVl-4. 964
(Air Shields) with a minute ventilation of 750 ml. on 40% oxygen. The lumbosacral area was shaved and the animal placed on a heating blanket. A rectal probe was inserted for temperature monitoring and a 5 French urethral catheter inserted into the bladder. Bladder pressures were measured via a Statham P23Dc pressure transducer and recorded on a Grass polygraph (Model 7D) after calibration and filling of the bladder with 10 cc of normal saline. A sterile approach was used to perform a lumbosacral laminectomy. The extradural fat was separated in the midline to expose the lowest lumbar and the sacral mixed spinal nerve roots. The segmental root level was identified anatomically since L 7 is larger than either L6 or 81 and there is a larger gap between L6 and L 7 than between L 7 and SL Confirmation of root level was done through electrical stimulation of the individual exposed root using bipolar platinum/iridium hook electrodes. The stimuli were current pulses of 0.1 ms duration at a rate of 10/sec. produced by a Grass stimulator (SD9) in series with a 10 K-ohm resistor. The amplitude of the current pulses was between 0.1 to 10 X 10-3 amps (current density 5 to 50 X 10-s amps/mm 2). In this phase of the study, eleven cats had a unilateral L 7 to 83 transection of the extradural roots distal to their dorsal root ganglia followed by an immedite L 7 to 81 anastomosis using conventional microsurgical techniques under a Zeiss operating microscope (Opmi 7). Six of the animals had a four to six mm. portion of the ipsilateral 82 root interposed in the connection. Each anastomosis was secured with three equally spaced 10-0 microsutures (Prolene, Ethicon on a BV75 needle) passed through the nerve root. Where practical, extradural fat was draped over the anastomosis. Postoperatively, a course of prophylactic antibiotics (ampicillin 6 mg./kg.) was administered intramuscularly for five days. The animals were given a period of seven months for axonal regeneration and urinary bladder reinnervation at which time they underwent reoperation to expose the previously anastomosed nerve root. Reoperation for cystometric and electrophysiologic data. Animal preparation for reoperation was similar to the initial surgery except that alpha chloralose (Calbiochem) prepared in polyethylene glycol 200 (60 mg./kg.) was used for anesthesia. The urethra was catheterized using a 5 French feeding tube
965
URINARY BLADDER REINNERVATION
modified to allow measurement of the urethral pressure as described by McGuire. 7 A lower midline abdominal incision was made and the ureters transected to allow bladder pressure measurements at constant volume. An umbilical tape was tied around the urethra at the bladder neck and four cm. distally to compartmentalize the proximal urethra for pressure measurements. The bladder was cannulated with a 5 French feeding tube and sutured in place. The animal was repositioned prone and the previous laminectomy site opened. Because of the scar tissue, delicate dissection was necessary to reexpose the anastomosed root and to expose the contralateral sacral roots. This was done with the aid of loupe magnification (2.5X). The pudendal nerves were also exposed in the ischiorectal fossae bilaterally and the animal placed in a stereotaxic frame (Kopf Instruments). Bladder and urethral catheters were connected to a Grass polygraph via pressure transducers as outlined above. Temperature and electrocardiogram were monitored continuously. Two pairs of Teca monopolar needle electrodes (MG25) were placed transcutaneously in the perianal muscles and transvaginally in the periurethral muscles for electromyogram (EMG) recordings of pelvic striated muscle activity (Teca-TE4). After stimulation studies on the S2 nerve root of the previously unoperated control side for baseline recordings, the control S2 and S3 nerve roots were transected. Next, an intradural approach was made to the previously operated and anstomosed root. The dorsal and ventral root components were individually dissected out and detached from the spinal cord and stimulation studies were carried out immediately. The dorsal and ventral roots were placed on bipolar platinum/iridium hook electrodes and covered with mineral oil at body temperature to minimize desiccation and stimulus spread. In addition, a piece of thin plastic sheet was positioned beneath the nerve suspended on the hook electrodes to further reduce current spread. The hook electrodes were accurately positioned with Narashige X-Y-Z ball-joint manipulators. Stimuli were delivered via the Grass stimulator as outlined previously. The EMG's were displayed on the Tectronix memory oscilloscope for verification; they were also connected to the input of a window discriminator (WPI, Model 121) which converted each waveform into a digital pulse. These pulses were sent to the Grass polygraph to compensate for the low pass filtering characteristics of the polygraph. Separate bladder and urethral responses and external sphincter EMG traces were recorded on the Grass polygraph (fig. 1). For compound action potential (CAP) recordings, the stimulating electrodes were placed on the dorsal and ventral nerve NORMAL ROOT STIMULATION
40
-
NORMAL S 1
L7-S1
---- ---- ----"\
\__
~
~
-----
_J
BLADDER PRESSURE
s2
~
L7-G-S 1
•
~
(cm H20)
20
-
PROXIMAL URETHRAL PRESSURE
------
+
-
-
(cm H20l
RAW
I
PERIURETHRAL EMG
\
10/s-
RATE
0
::·
L_
L
-----+--
--it-
½ '"_;.'
--
I- -
-
FIG. 1. Typical lower urinary tract responses to isolated root stimulation for normal 82, normal 81, L7-S1 anastomosis and L7-graft-Sl anastomosis. Time of stimulation was 10 seconds and for each root represents supramaximal response (varying voltage). Stimulated EMG activity is recorded as both raw data and as voltage proportional to frequency of EMG complexes.
roots just distal to their detachment from the spinal cord. The recording electrodes were placed on the ipsilateral pudendal nerve, which was severed distally just prior to this part of the study. The recording electrodes were connected to a preamplifier and amplifier and a dual beam oscilloscope (Tektronix). Photomicrorecords (Tektronix) of individual sweeps synchronized with the stimulus were obtained. Before stimulation, the nerve between the recording pair of electrodes was crushed to provide a monophasic CAP recording. In this study, two different CAPs were recorded at the pudendal nerve by electrically stimulating the ventral (orthodromic) and dorsal (antidromic) nerve root components separately. After both pudendal CAPs had been obtained, the recording electrodes were placed at the mixed root intraspinally immediately distal to the anastomosis. As with the pudendal nerve recordings, the intraspinal nerve was severed distal to the recording site and crushed between the two recording electrodes. Two additional CAPs were recorded at this site. In sum, four CAPs were recorded per nerve; two at the site of the pudendal nerve (dorsal and ventral components) and two intraspinally just distal to the anastomosis (dorsal and ventral components). Furthermore, for the normal control Sl and L 7 nerve roots four CAPs were recorded by the same stimulating/recording arrangements. Therefore, a comparison of the relative amounts of sensory (dorsal) and motor (ventral) regeneration between the anastomosed and control roots could be made for each animal (fig. 3). Quantification of experimental results. The cystometric and CAP data were quantified in the following way. Using a standard 10 second train of stimuli and a supramaximal current, the stimulated increase in bladder pressure recorded on the Grass polygraph was traced onto clear celluloid sheets and individual traces weighed on a Mettler balance. These weights provided a relative measure of the areas under the 10 second stimulated bladder pressure curves since the amplification, time axis, and celluloid sheets were the same in every experiment. This procedure is analogous to the standard area-under-the-curve measurements for quantifying CAP data. The CAP records, which were photographs of Tektronix memory oscilloscope tracings, were quantified in a similar fashion except correction was made when necessary for amplification and time base. Thus, weights of the cut-out CAP tracings were standardized to a single voltage and time base. All of the cystometric and CAP results are expressed in terms of these area-under-the-curve measurements (as in figs. 1 and 3). RESULTS
Cystometric data. Normal SJ and S2 bladder responses. Characteristic polygraph recordings documenting intraoperative bladder pressures at maximal stimulation of the control Sl and S2 roots are shown in figure 1. Immediately prior to these recordings the nerve roots had been detached and isolated from the spinal cord. Thus, in both normal and anastomosed roots bladder responses were elicited through nerve conduction along the spinal nerve root and not through alternative spinal and peripheral pathways. In figure 1, tracings from the normal Sl and S2 roots show a rise in bladder pressure with a concomitant decrease in proximal urethral pressure. The detrusor response obtained from S2 stimulation was larger than that obtained from Sl stimulation and was a consistent finding in all animals. However, some animals did not exhibit a bladder contraction upon Sl stimulation but did when S2 was stimulated. Nerve root stimulation also produced the usual increase in EMG activity which is opposite to that seen in normal micturition. EMG activity was noted consistently on Sl stimulation. Bladder responses after a lumbar to sacral root anastomosis. Stimulation of an L 7 root anastomosed to an Sl nerve root directly and through an S2 nerve graft is also shown in figure 1. Note that a sizable detrusor response was recorded with a concomitant electrically-stimulated increase in EMG activity. This record, and others like it, demonstrate significant regen-
966
VORSTMAN AND ASSOCIATES
eration and reinnervation of the urinary bladder after a nerve root crossover anastomosis. A comparison of the percentage detrusor responses for the anastomosed 17-Sl (with and without nerve grafts) and contralateral (normal; control) Sl nerve roots obtained in seven animals during maximal stimulation of the ,isolated roots detached from the spinal cord, is shown in figure 2. The cystometric data were quantified analogous to the standard areaunder-the-curve analysis. Note that two of the seven anastomosed nerves did not exhibit a response even though the control Sl did project to the bladder. The other five anastomosed roots showed responses of 40 to 140% of the control Sl detrusor response. The average size of the 17-Sl detrusor response (with and without nerve grafts) for all seven operated nerves was 56% compared to the size of the normal Sl detrusor response. This comparison suggests considerable lumbar root axonal regeneration and bladder reinnervation. Two animals died during anesthesia at the time of restudy. In an additional two animals, the normal Sl did not project to the bladder, and in those animals, stimulation of the anastomosed roots did not produce a bladder contraction. Compound action potential recordings. Typical CAP data obtained in an animal that showed bladder reinnervation after an L 7 to Sl anastomosis via an S2 nerve graft is shown in figure 3. Recordings were made just distal to the anastomosis (or distal to the dorsal root ganglion on the control side) and at the pudendal nerve. Monophasic CAPs are shown for the normal Sl root and the ipsilateral pudendal nerve. The size of the CAPs appears similar for both dorsal and ventral components of the extradural root and the pudendal nerve. However, recordings at the pudendal nerve are typically ten times smaller than those measured intraspinally due to the smaller size of the nerve. Also, because of an increased distance between the stimulating and recording electrodes, the pudendal nerve CAPs have an increased latency. Monophasic CAPs are also shown for the dorsal and ventral roots measured on the side of the nerve root anastomosis. The amplitude of the traces measured intraspinally are approximately half of that measured at the corresponding point for the contralateral normal Sl root. Similarly, the amplitude of the traces measured at the pudendal nerve on the side of the anastomosis is some four times smaller than those measured at the pudendal nerve on the control side indicating fewer active axons in the regenerated root than in the normal root. CAP recordings taken at the pudendal nerve and distal to ~
Cl)
'ii
OPERATED ROOT
NORMAL S1
V
D
AA Distal
Distal
D
V
A_Jl P,doodol
\
FIG. 3. Recordi~gs of normal Sl and operated root CAPs. Diagram exhibits typical dorsal and ventral root compound action potentials from normal Sl nerve root and from nerve root on opposite side of spinal cord in which proximal part of L7 had been anastomosed to distal end of Sl through nerve graft. For recordings, dura has been incised and retracted laterally to expose intradural roots on both sides of spinal cord (only one side shown). Roots were detached from cord immediately prior to stimulation in order to prevent indirect spinal pathways to bladder or recording sites. Stimulating electrodes (not shown) were positioned proximal to dorsal root ganglion. Recording electrodes (not shown) for "distal" CAP traces were placed just distal to anastomosis. For both operated L7-S1 and normal Sl roots this intraspinal location was approximately level with baseline of traces shown. Recording electrodes (not shown) for pudenda! CAP traces were positioned in ischiorectal fossae. In addition to transection of pudenda! distal to recording electrode, proximal branches of pudenda! nerves were cut to prevent any possible (reflexive) indirect neural activity from bladder. Therefore, CAP recordings reflect only degree of neural activity across anastomosis. CAP tracings shown distal to anastomosis were 2 msec in duration and 2 mV (normal Sl) and 0.8 mV (operated root) in amplitude. CAP tracings shown at pudenda! nerve are 8 msec in duration and 0.2 mV (normal Sl) and 0.05 mV (operated root) in amplitude.
~
0
z 0 0 a:
100
0.
g
GI
"'C g "'GI
50
a:
0
"'~
• C
'#
0 (7)
2
AVG.
L1 - S1
3
4
5
8
7
L1 - G - S1
FIG. 2. Graph compares percentage detrusor response (operated root/normal Sl) obtained during maximal stimulation of isolated operated roots detached from spinal cord for seven animals. Three animals had L 7 to Sl anastomosis while four animals had L 7 to Sl anastomosis through S2 nerve graft. Average size of reinnervated bladder contraction was 56% of normal Sl response.
the anatomosis during dorsal and ventral root stimulation were measured as percent size normalized to the contralateral Sl. Pudendal nerve CAPs (fig. 4). The percent size of the CAP recorded at the pudendal nerve on the side of the anastomosis was 67% for the dorsal root and 72% for the ventral root. Our findings also indicate that the normal Sl projects to the pudendal nerve consistently. However, the normal L 7 dorsal root contributes very little to the pudendal nerve while stimulation of the normal L 7 ventral root produced an average CAP recording some 30% of the normal Sl ventral root. The increase in CAP recordings at the pudendal nerve after an L7 to Sl root crossover anastomosis compared to pudendal CAPs from a normal L 7 root indicates considerable redirection of fibers. Intraspinal CAPs distal to the anastomosis (fig. 5). The percent size of the CAP recorded distal to the anastomosis was 29% for the dorsal root and 75% for the ventral root. These results indicate regeneration of both dorsal and ventral root axons although better for ventral root axons on the average. The normal 17 root did not project to the bladder. Although there was an increase in latency of the CAP measured distal to
URINARY BLADDER REINNERVATION 200
l
1501 Ji .E
25
I
*
E 100 0
~ I!.
<
0
0 N
50
00 "I#-
L7
l1-S1
S1
Dorsal Stimulation
L7
L7-S1
S1
Ventral Stimulation
CAP RECORDINGS FROM THE PUDENDAL NERVE
FIG. 4. Percent size of CAPs recorded at pudenda! nerve for anastomosed roots and L7 are graphed relative to normal S1 for both dorsal and ventral components. 81 response represents 100%. Stimulating
electrodes were placed on dorsal and ventral roots proximal to anastomosis and recording electrode was placed on pudenda! nerve in ischiorectal fossa. Data points are plotted for anastomoses without graft ( ·) and with nerve graft (*). Normal L7 dorsal root rarely projects to pudenda!.
'::. 100 (I)
0
E b 0
3 Cl..
u
50
0
(!)
N Cf)
.ft.
L7-S1
S1
DorsaJ Stimulation
L7-S1
S1
Ventral Stimulation
CAP RECORDINGS FROM THE DIST AL ROOT FIG. 5. Percent size of CAPs recorded intraspinally distal to anas-
tomoses for operated roots are graphed relative to normal 81 for both dorsal and ventral components. Sl response represents 100%. Stimulating electrodes were placed on dorsal and ventral roots proximal to anastomosis and recording electrode was placed on mixed root intraspinally distal to anastomosis. Data points are plotted for anastomoses without graft (.) and with nerve graft (*). the anastomosis and at the pudendal of the operated side when compared to the control side, the relatively large size of the CAPs indicates, as before, substantial axonal regeneration. DISCUSSION
This study has shown that the axons of a suprasacral mixed root (L 7) when anastomosed to a sacral mixed root (Sl) can regenerate and recentralize the unilaterally decentralized bladder in the cat. Furthermore, this is the first report to show that bladder reinnervation can occur through a nerve graft interposed between adjacent nerve roots. The evidence for successful regeneration and reinnervation was derived from the recording of bladder contractions and striated muscle activity from the external sphincter during stimulation of the anastomosed roots
967
after detachment from the spinal cord (figs. 1, 2). Compound action potential (CAPs) recording from the ipsilateral pudendal nerve and those recorded immediately distal to the anastomosis confirmed both dorsal and ventral root axonal regeneration (figs. 3, 4, 5). These data were compared to similar data obtained from the contralateral non-operated (control) roots. In addition, we have presented evidence for significant redirection of axons from a lumbar root to the bladder after nerve root crossover anastomosis (fig. 5). In this study, quantitative measurement of bladder reinnervation has supported previous qualitative measurement examining return of the micturition reflex. 6 ' 8 •9 However, as in the other studies we have shown functional regeneration but not necessarily return of useful bladder function. Such a study may require bilateral nerve root surgery and an analysis of the storage/voiding cycle. A reproducible bladder contraction was noted in five of seven animals with anastomosed L 7-Sl roots (with and without nerve grafts) when there was a contraction generated by stimulation of the control Sl root. In an additional two animals the normal Sl did not project to the bladder, and in those animals stimulation of the anastomosed root also did not produce a bladder contraction. However, CAPs and EMG activity were recorded indicating regeneration of axons through the anastomosis. Furthermore, there was little difference in the size of the detrusor response between roots connected with and without a nerve graft. A factor that may have influenced the success of bladder reinnervation through a nerve graft was the use of a spinal nerve root as a graft. Segments of adjacent nerve roots used as grafts in a nerve root anastomosis may approximate each other better because of size, fascicular pattern and endoneurial tube diameter. The achievement of bladder reinnervation through such a technique suggests that the use of nerve grafts to bridge gaps in crossover nerve anastomoses can be helpful in bypassing a spinal cord lesion. The bladder responses to stimulation of the previously anastomosed roots generally were reproducible and larger than expected. However, the size of the evoked bladder contraction was smaller than that of the normal SL In fact, the average size of the detrusor response through the anastomosed roots was 56% of the normal Sl root although one L 7-Sl anastomosis via a nerve graft gave a larger bladder contraction than the contralateral normal SL Reasons for this anomalous result may have been an asymmetrical projection of the Sl axons to the bladder or an increased number of axons to the detrusor because of axon sprouting. 10 To enhance the possibility of an adequate response to stimulation in the reinnervated bladder, the root anastomosis should involve the dominant root to the bladder. With the increased projection of axons from the dominant root to the bladder, reinnervation a suprasacral root would be more likely and return of function may be facilitated. In other studies, the return of bladder contractility has been generally successful. 3 · 6 Microsurgical repair of nerve roots has been reported previously but the usefulness of this technique is unclear since the return of the micturition reflex has been documented after simple approximation of nerve ends using a millipore tubulation technique. 8 The time for return of the micturition reflex in these animals who had ventral root only anastomoses varied between four and seven months. Present techniques of nerve anastomosis may be the primary limiting factor in the achievement of useful function. In view of this problem, a new method of reconnecting peripheral nerves resulting in improved functional return has recently been investigated and may eventually be adapted for nerve root and spinal cord surgery. 11 An evaluation of efferent supply (predominantly via the ventral roots) to the bladder and external urethral sphincter was possible through the cystometric and EMG recordings. 12 However, stimulation studies investigating efferent responses provide no indication of whether dorsal root (sensory) axonal
968
VORSTMAN AND ASSOCIATES
regeneration had occurred. Therefore, dorsal root CAP data provide the only measure of sensory axonal regeneration across the anastomoses while ventral root axonal regeneration was documented by both cystometric and CAP data. CAPs were documented across all the reconstructed roots and at the ipsilateral pudendal nerve. The relatively large size of the CAPs recorded from both the dorsal and ventral root components indicates substantial axonal regeneration (fig. 3). In fact, the percent size of the CAPs recorded at the pudendal nerve after an L7-Sl anastomosis was similar for both dorsal and ventral roots. Our studies have also shown that the normal contralateral L 7 root has a limited projection to the pudendal nerve. However, the CAPs recorded at the pudendal nerve on the side of the anastomosed root were much larger, indicating significant redirection of fibers. Despite these encouraging results, CAP data have limited usefulness. Although CAPs give an indication of axonal regeneration with respect to the number of axons crossing a repair site, they cannot predict the number of proximal axons with distal connections. Similarly, no definitive evidence for functional reinnervation can be derived from CAP data. 10 Previous studies have suggested that L7 (somatic) axons can reinnervate the parasympathetically denervated bladder after nerve crossover experiments.a Recent investigations have also provided evidence that functional reinnervation of other parasympathetic ganglia by somatic axons can occur through a fraction of the synapses riormally found. 1a These observations suggest considerable redundancy in normal innervation and that return of function with somatic axons in a previously autonomically innervated structure may be possible. The mechanism of urinary bladder reinnervation by somatic axons is not known. Possible explanations include somatic axonal reinnervation of intramural ganglia and bladder smooth muscle or reinnervation of the ganglia alone as the intramural ganglia of the detrusor are merely decentralized after sacral root transection. However, temporary detrusor muscle denervation may also occur secondary to transaxonal degeneration of post-ganglionic neurons but axonal regeneration usually begins within four weeks. 14 Therefore, the process of urinary bladder reinnervation in the study outlined appears to involve somatic axonal reinnervation of intramural ganglia while the bladder smooth muscle is probably reinnervated by post ganglionic cholinergic and adrenergic axons. Urinary bladder reinnervation may also be influenced by changes in the sympathetic supply to the bladder as a consequence of parasympathetic denervation. After such denervation, a change in detrusor response can be detected at six weeks and with this change alpha adrenoreceptor function appears in the nerve terminals rather than the normal beta adrenoreceptor activity. 15 The sprouting of adrenergic nerves in the bladder and change in receptor status may be a direct result of the parasympathetic decentralization. How this local sprouting by adrenergic nerves affects subsequent reinnervation by regenerating lumbar axons is not known. In addition, sex steroids have been shown to play a role in the regulation of the density of neurotransmitter receptors and this may influence the degree of reinnervation achieved in the bladders of female individuals compared to male individuals. 16 The mechanisms of the micturition reflex are complex and controversial. However, an intact pontine micturition center appears to be important and recovery of the bladder's storage/ voiding cycle via nerve root crossover anastomoses after decentralization demands a modifiable or plastic nervous system. 17 Recently, evidence supporting the ability for synapse turnover and neuronal plasticity after injury in the mammalian nervous system was reviewed. 18· 19 Although axonal regeneration rates of one to five mm./24 hours (for sensory fibers after suture) have been documented, the time required for neuroplasticity or the development of these alternative central nervous system connections is not known. 20 Similarly, the number of appropri-
ately connected regenerated axons necessary for restoration of useful bladder function may be far fewer than anticipated because of the considerable redundance in detrusor innervation. 21 The postulate that return of bladder function after extensive neural injury is possible through collateral sprouting, sympathetic pathways or detrusor contractions initiated by intramural ganglia, appears to be no longer tenable. 8 Urinary bladder reinnervation (or reinnervation of the intramural ganglia) through nerve crossover surgery is feasible in the cat and, as observed by others, an increase in the neurologic deficit after such a technique is usually not significant clinically.2·a However, restoration of useful urinary bladder function by establishing alternative pathways for suprasacral bladder control remains to be confirmed. Presently we are performing studies examining the return of useful bladder function after crossover nerve root surgery. Other experiments using enzyme tracer techniques and investigations of somatosensory responses will also allow a better understanding of bladder function after nerve injury and reinnervation. Information from such investigations may then allow the use of similar nerve crossover techniques in selected patients with spinal cord injury or spina bifida to restore functional micturition. 22 The appropriate timing of such surgical intervention is not known yet. Furthermore, a factor contributing to the successful return of functional micturition in these patients after nerve crossover surgery will be the degree to which detrusor viability has been maintained. Detrusor fibrosis, because of chronic inflammation, could compromise return of useful bladder function because of myogenic rather than neurogenic factors.
Acknowledgments. The authors are grateful for the support and encouragement extended by Edward McGuire, M.D., Luis de Medinaceli, M.D. and William de Groat, Ph.D. REFERENCES
1. Vorstman, B., Schlossberg, S. and Kass, L.: Can severe spinal nerve injury be repaired? JAMA, 254: 55, 1985. 2. Kilvington, B.: An investigation on the regeneration of nerves with regard to surgical treatment of certain paralyses. Br. Med. J., 1: 988, 1907. 3. Sundin, T.: Reinnervation of the urinary bladder. An experimental study in cats. Scand. J. Urol. Nephrol. (Suppl.), 17: 3, 1972. 4. Carlsson, C. A. and Sundin, T.: Reconstruction of afferent and efferent nervous pathways to the urinary bladder in two paraplegic patients. Spine, 5: 37, 1980. 5. Carlsson, C. A. and Sundin, T.: Forefront: preliminary report. Reconstruction of efferent pathways to the urinary bladder in a paraplegic child. Review Surg., 24: 73, 1967. 6. Conzen, M. A. and Sollmann, H.: Reinnervation of the urinary bladder after microsurgical reconstruction of transected caudal fibers: an experimental study in pigs. Urol. Res., 10: 141, 1982. 7. McGuire, E. J. and Morrissey, S. G.: The development of reflex bladder activity following spinal cord injury in cats and a method to control it. Neurourol. Urodynam., 1: 211, 1982. 8. Carlsson, C. A. and Sundin, T.: Reconstruction of severed ventral roots innervating the urinary bladder: an experimental study in cats. Scand. J. Urol. Nephrol., 2: 199, 1968. 9. Sundin, T. and Carlsson, C. A.: Reconstruction of severed dorsal roots innervating the urinary bladder: an experimental study in cats. II. Regeneration studies. Scand. J. Urol. Nephrol., 6: 185, 1972. 10. Rosen, J.M. and Jewett, D. L.: Physiological methods of evaluating experimental nerve repairs. In: Nerve Repair and Regeneration. Its Clinical and Experimental Basis. Edited by D. L. Jewett and H. R. McCarroll. St. Louis: The C.V. Mosby Co., ch. 15, p. 150, 1979. 11. de Medinaceli, L., Wyatt, R. J. and Freed, W. J.: Peripheral nerve reconnection: mechanical, thermal and ionic conditions that promote the return of function. Exp. Neurol., 81: 469, 1983. 12. Coggeshall, R. E. and Ito, H.: Sensory fibers in ventral roots L7 and 81 in the cat. J. Physiol., 267: 215, 1977.
969 13. Proctor, Vv., Roper, S. and Taylor, B.: Somatic axons can innervate the autonomic neurones in the frog heart. J. Physiol., 326: 173, 1982. 14. Elbadawi, A., Atta, M.A. and Franck, J. I.: Intrinsic neuromuscular defects in the neurogenic bladder. I. Short-term ultrastructural changes in muscular innervation of the decentralized feline bladder base following unilateral sacral ventral rhizotomy. Neurourol. Urodynam., 3: 93, 1984. 15. Sundin, T. and Dahlstrom, A.: The sympathetic innervation of the urinary bladder and urethra in the normal state and after parasympathetic denervation at the spinal root level. Scand. J. Urol. Nephrol., 7: 131, 1973. 16. Levin, R. M., Jacobowitz, D. and Wein, A. J.: Autonomic innervation of rabbit urinary bladder following estrogen administration. Urology, 17: 449, 1981. 17. de Groat, W. C.: Nervous control of the urinary bladder of the cat. Brain Res., 87: 201, 1975.
18. Freed, W. J., de Medinaceli, L. and Wyatt, R. J.: Promoting functional plasticity in the damaged nervous system. Science, 227: 1544, 1985. 19. Cotman, C. W. and Nieto-Sampedro, M.: Cell biology of synaptic plasticity. Science, 225: 1287, 1984. 20. Sunderland, S.: Nerves and Nerve Injuries. Churchill Livingston, New York, Second Edition, p. 121, 1978. 21. Gero!, A. Y., Seymour, R. J., Pava, A. A., Robertson, J. W., Boyesen, S., Callan, D. and Campbell, J.: Evidence of reserve in the innervation of the urinary bladder of cats: a preliminary study directed at improving the therapeutic approach to bladder dysfunction in man following cauda equina injury. Surg. Forum, 6: 499, 1955. 22. Vorstman, B., Schlossberg, S., Kass, L., Devine, C. J. Jr. and Horton, C. E.: Spinal nerve root surgery for urinary bladder reinnervation. Neurourol. Urodynam., 5: 327, 1986.
THE JOURNAL OF UROLOGY
Copyright© 1986 by The Williams & Wilkins Co.
URINARY BLADDER REINNERVATION BERT VORSTMAN,* STEVEN M. SCHLOSSBERG, LEONARD KASS
AND
CHARLES J. DEVINE, JR.
From the Departments of Urology and Physiology, Eastern Virginia Medical School, Norfolk, Virginia
ABSTRACT
The ability of mixed spinal nerve roots to regenerate and reinnervate the urinary bladder was examined in young adult female cats. Using microsurgical technique, a unilateral extradural spinal nerve root anastomosis of a lumbar (L 7) to a sacral root (Sl) either with or without a nerve graft was performed. Remaining ipsilateral sacral roots were transected. The contralateral normal sacral roots remained intact and allowed the animals adequate urination during the period necessary for axonal regeneration. At the time of restudy seven months later, stimulation of the anastomosed nerve root proximal to the anastomosis (isolated from the spinal cord) elicited a bladder contraction. Significant lumbar axonal regeneration was substantiated by compound action potentials recorded across the anastomosis. In addition, redirection of axons from a lumbar to a sacral distribution was demonstrated. The contralateral normal sacral roots provided control cystometric and electrophysiological data against which responses from the previously anastomosed nerve roots were compared. In conclusion, significant bladder reinnervation can occur after an anastomosis of a lumbar and sacral root with or without a nerve graft. This technique, or variations thereof, may have a clinical role in selected patients with neurogenic bladder dysfunction to reinnervate the bladder and restore central control. Current treatment of the decentralized bladder has decreased the morbidity and mortality associated with the condition but has not realized functional micturition. 1 In fact, research into restoring central control of the neurogenic bladder has been limited. Attempts at urinary bladder reinnervation have been described and, although first conceptualized in 1907, few studies have examined cystometric and compound action potential data to analyze systematically urinary bladder reinnervation. 2 Both intradural and extradural nerve root crossover surgical techniques have been performed in animal models and a few studies on paraplegic and spina bifida patients have also been reported.2-5 Recently, an experimental study was described that documented the return of the micturition reflex after an intradural microsurgical reconstruction of sacral motor and sensory roots. 6 Urinary bladder reinnervation or recentralization through the use of mixed spinal nerve roots and nerve grafts has not been reported previously. Nerve grafts would likely be necessary for bypassing an injured segment of the spinal cord for reinnervation procedures in humans because of the distance to be bridged between functional nerve roots above the injured segment and sacral roots below. In the present work, the following questions were investigated: a) whether electrophysiologic and cystometric data would indicate axonal regeneration and bladder reinnervation after a mixed suprasacral nerve root (L 7) to mixed sacral root (81) anastomosis; b) whether similar data might be obtained after a nerve root anastomosis via a nerve graft; and c) whether a lumbar to sacral mixed root anastomosis would provide for redirection of axons. MATERIALS AND METHODS
Initial surgery. Eleven adult female cats (two to three kg.) were anesthetized with intramuscular ketamine (25 mg./kg.). An intravenous line was inserted for administration of normal saline, ampicillin and incremental doses of phenobarbital. The animals were intubated and connected to a volume ventilator Accepted for publication May 9, 1986. * Requests for reprints: Department of Urology, University of Miami School of Medicine, Miami, FL 33101. Supported by American Paralysis Association grant BWVl-4. 964
(Air Shields) with a minute ventilation of 750 ml. on 40% oxygen. The lumbosacral area was shaved and the animal placed on a heating blanket. A rectal probe was inserted for temperature monitoring and a 5 French urethral catheter inserted into the bladder. Bladder pressures were measured via a Statham P23Dc pressure transducer and recorded on a Grass polygraph (Model 7D) after calibration and filling of the bladder with 10 cc of normal saline. A sterile approach was used to perform a lumbosacral laminectomy. The extradural fat was separated in the midline to expose the lowest lumbar and the sacral mixed spinal nerve roots. The segmental root level was identified anatomically since L 7 is larger than either L6 or 81 and there is a larger gap between L6 and L 7 than between L 7 and SL Confirmation of root level was done through electrical stimulation of the individual exposed root using bipolar platinum/iridium hook electrodes. The stimuli were current pulses of 0.1 ms duration at a rate of 10/sec. produced by a Grass stimulator (SD9) in series with a 10 K-ohm resistor. The amplitude of the current pulses was between 0.1 to 10 X 10-3 amps (current density 5 to 50 X 10-s amps/mm 2). In this phase of the study, eleven cats had a unilateral L 7 to 83 transection of the extradural roots distal to their dorsal root ganglia followed by an immedite L 7 to 81 anastomosis using conventional microsurgical techniques under a Zeiss operating microscope (Opmi 7). Six of the animals had a four to six mm. portion of the ipsilateral 82 root interposed in the connection. Each anastomosis was secured with three equally spaced 10-0 microsutures (Prolene, Ethicon on a BV75 needle) passed through the nerve root. Where practical, extradural fat was draped over the anastomosis. Postoperatively, a course of prophylactic antibiotics (ampicillin 6 mg./kg.) was administered intramuscularly for five days. The animals were given a period of seven months for axonal regeneration and urinary bladder reinnervation at which time they underwent reoperation to expose the previously anastomosed nerve root. Reoperation for cystometric and electrophysiologic data. Animal preparation for reoperation was similar to the initial surgery except that alpha chloralose (Calbiochem) prepared in polyethylene glycol 200 (60 mg./kg.) was used for anesthesia. The urethra was catheterized using a 5 French feeding tube
965
URINARY BLADDER REINNERVATION
modified to allow measurement of the urethral pressure as described by McGuire. 7 A lower midline abdominal incision was made and the ureters transected to allow bladder pressure measurements at constant volume. An umbilical tape was tied around the urethra at the bladder neck and four cm. distally to compartmentalize the proximal urethra for pressure measurements. The bladder was cannulated with a 5 French feeding tube and sutured in place. The animal was repositioned prone and the previous laminectomy site opened. Because of the scar tissue, delicate dissection was necessary to reexpose the anastomosed root and to expose the contralateral sacral roots. This was done with the aid of loupe magnification (2.5X). The pudendal nerves were also exposed in the ischiorectal fossae bilaterally and the animal placed in a stereotaxic frame (Kopf Instruments). Bladder and urethral catheters were connected to a Grass polygraph via pressure transducers as outlined above. Temperature and electrocardiogram were monitored continuously. Two pairs of Teca monopolar needle electrodes (MG25) were placed transcutaneously in the perianal muscles and transvaginally in the periurethral muscles for electromyogram (EMG) recordings of pelvic striated muscle activity (Teca-TE4). After stimulation studies on the S2 nerve root of the previously unoperated control side for baseline recordings, the control S2 and S3 nerve roots were transected. Next, an intradural approach was made to the previously operated and anstomosed root. The dorsal and ventral root components were individually dissected out and detached from the spinal cord and stimulation studies were carried out immediately. The dorsal and ventral roots were placed on bipolar platinum/iridium hook electrodes and covered with mineral oil at body temperature to minimize desiccation and stimulus spread. In addition, a piece of thin plastic sheet was positioned beneath the nerve suspended on the hook electrodes to further reduce current spread. The hook electrodes were accurately positioned with Narashige X-Y-Z ball-joint manipulators. Stimuli were delivered via the Grass stimulator as outlined previously. The EMG's were displayed on the Tectronix memory oscilloscope for verification; they were also connected to the input of a window discriminator (WPI, Model 121) which converted each waveform into a digital pulse. These pulses were sent to the Grass polygraph to compensate for the low pass filtering characteristics of the polygraph. Separate bladder and urethral responses and external sphincter EMG traces were recorded on the Grass polygraph (fig. 1). For compound action potential (CAP) recordings, the stimulating electrodes were placed on the dorsal and ventral nerve NORMAL ROOT STIMULATION
40
-
NORMAL S 1
L7-S1
---- ---- ----"\
\__
~
~
-----
_J
BLADDER PRESSURE
s2
~
L7-G-S 1
•
~
(cm H20)
20
-
PROXIMAL URETHRAL PRESSURE
------
+
-
-
(cm H20l
RAW
I
PERIURETHRAL EMG
\
10/s-
RATE
0
::·
L_
L
-----+--
--it-
½ '"_;.'
--
I- -
-
FIG. 1. Typical lower urinary tract responses to isolated root stimulation for normal 82, normal 81, L7-S1 anastomosis and L7-graft-Sl anastomosis. Time of stimulation was 10 seconds and for each root represents supramaximal response (varying voltage). Stimulated EMG activity is recorded as both raw data and as voltage proportional to frequency of EMG complexes.
roots just distal to their detachment from the spinal cord. The recording electrodes were placed on the ipsilateral pudendal nerve, which was severed distally just prior to this part of the study. The recording electrodes were connected to a preamplifier and amplifier and a dual beam oscilloscope (Tektronix). Photomicrorecords (Tektronix) of individual sweeps synchronized with the stimulus were obtained. Before stimulation, the nerve between the recording pair of electrodes was crushed to provide a monophasic CAP recording. In this study, two different CAPs were recorded at the pudendal nerve by electrically stimulating the ventral (orthodromic) and dorsal (antidromic) nerve root components separately. After both pudendal CAPs had been obtained, the recording electrodes were placed at the mixed root intraspinally immediately distal to the anastomosis. As with the pudendal nerve recordings, the intraspinal nerve was severed distal to the recording site and crushed between the two recording electrodes. Two additional CAPs were recorded at this site. In sum, four CAPs were recorded per nerve; two at the site of the pudendal nerve (dorsal and ventral components) and two intraspinally just distal to the anastomosis (dorsal and ventral components). Furthermore, for the normal control Sl and L 7 nerve roots four CAPs were recorded by the same stimulating/recording arrangements. Therefore, a comparison of the relative amounts of sensory (dorsal) and motor (ventral) regeneration between the anastomosed and control roots could be made for each animal (fig. 3). Quantification of experimental results. The cystometric and CAP data were quantified in the following way. Using a standard 10 second train of stimuli and a supramaximal current, the stimulated increase in bladder pressure recorded on the Grass polygraph was traced onto clear celluloid sheets and individual traces weighed on a Mettler balance. These weights provided a relative measure of the areas under the 10 second stimulated bladder pressure curves since the amplification, time axis, and celluloid sheets were the same in every experiment. This procedure is analogous to the standard area-under-the-curve measurements for quantifying CAP data. The CAP records, which were photographs of Tektronix memory oscilloscope tracings, were quantified in a similar fashion except correction was made when necessary for amplification and time base. Thus, weights of the cut-out CAP tracings were standardized to a single voltage and time base. All of the cystometric and CAP results are expressed in terms of these area-under-the-curve measurements (as in figs. 1 and 3). RESULTS
Cystometric data. Normal SJ and S2 bladder responses. Characteristic polygraph recordings documenting intraoperative bladder pressures at maximal stimulation of the control Sl and S2 roots are shown in figure 1. Immediately prior to these recordings the nerve roots had been detached and isolated from the spinal cord. Thus, in both normal and anastomosed roots bladder responses were elicited through nerve conduction along the spinal nerve root and not through alternative spinal and peripheral pathways. In figure 1, tracings from the normal Sl and S2 roots show a rise in bladder pressure with a concomitant decrease in proximal urethral pressure. The detrusor response obtained from S2 stimulation was larger than that obtained from Sl stimulation and was a consistent finding in all animals. However, some animals did not exhibit a bladder contraction upon Sl stimulation but did when S2 was stimulated. Nerve root stimulation also produced the usual increase in EMG activity which is opposite to that seen in normal micturition. EMG activity was noted consistently on Sl stimulation. Bladder responses after a lumbar to sacral root anastomosis. Stimulation of an L 7 root anastomosed to an Sl nerve root directly and through an S2 nerve graft is also shown in figure 1. Note that a sizable detrusor response was recorded with a concomitant electrically-stimulated increase in EMG activity. This record, and others like it, demonstrate significant regen-
966
VORSTMAN AND ASSOCIATES
eration and reinnervation of the urinary bladder after a nerve root crossover anastomosis. A comparison of the percentage detrusor responses for the anastomosed 17-Sl (with and without nerve grafts) and contralateral (normal; control) Sl nerve roots obtained in seven animals during maximal stimulation of the ,isolated roots detached from the spinal cord, is shown in figure 2. The cystometric data were quantified analogous to the standard areaunder-the-curve analysis. Note that two of the seven anastomosed nerves did not exhibit a response even though the control Sl did project to the bladder. The other five anastomosed roots showed responses of 40 to 140% of the control Sl detrusor response. The average size of the 17-Sl detrusor response (with and without nerve grafts) for all seven operated nerves was 56% compared to the size of the normal Sl detrusor response. This comparison suggests considerable lumbar root axonal regeneration and bladder reinnervation. Two animals died during anesthesia at the time of restudy. In an additional two animals, the normal Sl did not project to the bladder, and in those animals, stimulation of the anastomosed roots did not produce a bladder contraction. Compound action potential recordings. Typical CAP data obtained in an animal that showed bladder reinnervation after an L 7 to Sl anastomosis via an S2 nerve graft is shown in figure 3. Recordings were made just distal to the anastomosis (or distal to the dorsal root ganglion on the control side) and at the pudendal nerve. Monophasic CAPs are shown for the normal Sl root and the ipsilateral pudendal nerve. The size of the CAPs appears similar for both dorsal and ventral components of the extradural root and the pudendal nerve. However, recordings at the pudendal nerve are typically ten times smaller than those measured intraspinally due to the smaller size of the nerve. Also, because of an increased distance between the stimulating and recording electrodes, the pudendal nerve CAPs have an increased latency. Monophasic CAPs are also shown for the dorsal and ventral roots measured on the side of the nerve root anastomosis. The amplitude of the traces measured intraspinally are approximately half of that measured at the corresponding point for the contralateral normal Sl root. Similarly, the amplitude of the traces measured at the pudendal nerve on the side of the anastomosis is some four times smaller than those measured at the pudendal nerve on the control side indicating fewer active axons in the regenerated root than in the normal root. CAP recordings taken at the pudendal nerve and distal to ~
Cl)
'ii
OPERATED ROOT
NORMAL S1
V
D
AA Distal
Distal
D
V
A_Jl P,doodol
\
FIG. 3. Recordi~gs of normal Sl and operated root CAPs. Diagram exhibits typical dorsal and ventral root compound action potentials from normal Sl nerve root and from nerve root on opposite side of spinal cord in which proximal part of L7 had been anastomosed to distal end of Sl through nerve graft. For recordings, dura has been incised and retracted laterally to expose intradural roots on both sides of spinal cord (only one side shown). Roots were detached from cord immediately prior to stimulation in order to prevent indirect spinal pathways to bladder or recording sites. Stimulating electrodes (not shown) were positioned proximal to dorsal root ganglion. Recording electrodes (not shown) for "distal" CAP traces were placed just distal to anastomosis. For both operated L7-S1 and normal Sl roots this intraspinal location was approximately level with baseline of traces shown. Recording electrodes (not shown) for pudenda! CAP traces were positioned in ischiorectal fossae. In addition to transection of pudenda! distal to recording electrode, proximal branches of pudenda! nerves were cut to prevent any possible (reflexive) indirect neural activity from bladder. Therefore, CAP recordings reflect only degree of neural activity across anastomosis. CAP tracings shown distal to anastomosis were 2 msec in duration and 2 mV (normal Sl) and 0.8 mV (operated root) in amplitude. CAP tracings shown at pudenda! nerve are 8 msec in duration and 0.2 mV (normal Sl) and 0.05 mV (operated root) in amplitude.
~
0
z 0 0 a:
100
0.
g
GI
"'C g "'GI
50
a:
0
"'~
• C
'#
0 (7)
2
AVG.
L1 - S1
3
4
5
8
7
L1 - G - S1
FIG. 2. Graph compares percentage detrusor response (operated root/normal Sl) obtained during maximal stimulation of isolated operated roots detached from spinal cord for seven animals. Three animals had L 7 to Sl anastomosis while four animals had L 7 to Sl anastomosis through S2 nerve graft. Average size of reinnervated bladder contraction was 56% of normal Sl response.
the anatomosis during dorsal and ventral root stimulation were measured as percent size normalized to the contralateral Sl. Pudendal nerve CAPs (fig. 4). The percent size of the CAP recorded at the pudendal nerve on the side of the anastomosis was 67% for the dorsal root and 72% for the ventral root. Our findings also indicate that the normal Sl projects to the pudendal nerve consistently. However, the normal L 7 dorsal root contributes very little to the pudendal nerve while stimulation of the normal L 7 ventral root produced an average CAP recording some 30% of the normal Sl ventral root. The increase in CAP recordings at the pudendal nerve after an L7 to Sl root crossover anastomosis compared to pudendal CAPs from a normal L 7 root indicates considerable redirection of fibers. Intraspinal CAPs distal to the anastomosis (fig. 5). The percent size of the CAP recorded distal to the anastomosis was 29% for the dorsal root and 75% for the ventral root. These results indicate regeneration of both dorsal and ventral root axons although better for ventral root axons on the average. The normal 17 root did not project to the bladder. Although there was an increase in latency of the CAP measured distal to
URINARY BLADDER REINNERVATION 200
l
1501 Ji .E
25
I
*
E 100 0
~ I!.
<
0
0 N
50
00 "I#-
L7
l1-S1
S1
Dorsal Stimulation
L7
L7-S1
S1
Ventral Stimulation
CAP RECORDINGS FROM THE PUDENDAL NERVE
FIG. 4. Percent size of CAPs recorded at pudenda! nerve for anastomosed roots and L7 are graphed relative to normal S1 for both dorsal and ventral components. 81 response represents 100%. Stimulating
electrodes were placed on dorsal and ventral roots proximal to anastomosis and recording electrode was placed on pudenda! nerve in ischiorectal fossa. Data points are plotted for anastomoses without graft ( ·) and with nerve graft (*). Normal L7 dorsal root rarely projects to pudenda!.
'::. 100 (I)
0
E b 0
3 Cl..
u
50
0
(!)
N Cf)
.ft.
L7-S1
S1
DorsaJ Stimulation
L7-S1
S1
Ventral Stimulation
CAP RECORDINGS FROM THE DIST AL ROOT FIG. 5. Percent size of CAPs recorded intraspinally distal to anas-
tomoses for operated roots are graphed relative to normal 81 for both dorsal and ventral components. Sl response represents 100%. Stimulating electrodes were placed on dorsal and ventral roots proximal to anastomosis and recording electrode was placed on mixed root intraspinally distal to anastomosis. Data points are plotted for anastomoses without graft (.) and with nerve graft (*). the anastomosis and at the pudendal of the operated side when compared to the control side, the relatively large size of the CAPs indicates, as before, substantial axonal regeneration. DISCUSSION
This study has shown that the axons of a suprasacral mixed root (L 7) when anastomosed to a sacral mixed root (Sl) can regenerate and recentralize the unilaterally decentralized bladder in the cat. Furthermore, this is the first report to show that bladder reinnervation can occur through a nerve graft interposed between adjacent nerve roots. The evidence for successful regeneration and reinnervation was derived from the recording of bladder contractions and striated muscle activity from the external sphincter during stimulation of the anastomosed roots
967
after detachment from the spinal cord (figs. 1, 2). Compound action potential (CAPs) recording from the ipsilateral pudendal nerve and those recorded immediately distal to the anastomosis confirmed both dorsal and ventral root axonal regeneration (figs. 3, 4, 5). These data were compared to similar data obtained from the contralateral non-operated (control) roots. In addition, we have presented evidence for significant redirection of axons from a lumbar root to the bladder after nerve root crossover anastomosis (fig. 5). In this study, quantitative measurement of bladder reinnervation has supported previous qualitative measurement examining return of the micturition reflex. 6 ' 8 •9 However, as in the other studies we have shown functional regeneration but not necessarily return of useful bladder function. Such a study may require bilateral nerve root surgery and an analysis of the storage/voiding cycle. A reproducible bladder contraction was noted in five of seven animals with anastomosed L 7-Sl roots (with and without nerve grafts) when there was a contraction generated by stimulation of the control Sl root. In an additional two animals the normal Sl did not project to the bladder, and in those animals stimulation of the anastomosed root also did not produce a bladder contraction. However, CAPs and EMG activity were recorded indicating regeneration of axons through the anastomosis. Furthermore, there was little difference in the size of the detrusor response between roots connected with and without a nerve graft. A factor that may have influenced the success of bladder reinnervation through a nerve graft was the use of a spinal nerve root as a graft. Segments of adjacent nerve roots used as grafts in a nerve root anastomosis may approximate each other better because of size, fascicular pattern and endoneurial tube diameter. The achievement of bladder reinnervation through such a technique suggests that the use of nerve grafts to bridge gaps in crossover nerve anastomoses can be helpful in bypassing a spinal cord lesion. The bladder responses to stimulation of the previously anastomosed roots generally were reproducible and larger than expected. However, the size of the evoked bladder contraction was smaller than that of the normal SL In fact, the average size of the detrusor response through the anastomosed roots was 56% of the normal Sl root although one L 7-Sl anastomosis via a nerve graft gave a larger bladder contraction than the contralateral normal SL Reasons for this anomalous result may have been an asymmetrical projection of the Sl axons to the bladder or an increased number of axons to the detrusor because of axon sprouting. 10 To enhance the possibility of an adequate response to stimulation in the reinnervated bladder, the root anastomosis should involve the dominant root to the bladder. With the increased projection of axons from the dominant root to the bladder, reinnervation a suprasacral root would be more likely and return of function may be facilitated. In other studies, the return of bladder contractility has been generally successful. 3 · 6 Microsurgical repair of nerve roots has been reported previously but the usefulness of this technique is unclear since the return of the micturition reflex has been documented after simple approximation of nerve ends using a millipore tubulation technique. 8 The time for return of the micturition reflex in these animals who had ventral root only anastomoses varied between four and seven months. Present techniques of nerve anastomosis may be the primary limiting factor in the achievement of useful function. In view of this problem, a new method of reconnecting peripheral nerves resulting in improved functional return has recently been investigated and may eventually be adapted for nerve root and spinal cord surgery. 11 An evaluation of efferent supply (predominantly via the ventral roots) to the bladder and external urethral sphincter was possible through the cystometric and EMG recordings. 12 However, stimulation studies investigating efferent responses provide no indication of whether dorsal root (sensory) axonal
968
VORSTMAN AND ASSOCIATES
regeneration had occurred. Therefore, dorsal root CAP data provide the only measure of sensory axonal regeneration across the anastomoses while ventral root axonal regeneration was documented by both cystometric and CAP data. CAPs were documented across all the reconstructed roots and at the ipsilateral pudendal nerve. The relatively large size of the CAPs recorded from both the dorsal and ventral root components indicates substantial axonal regeneration (fig. 3). In fact, the percent size of the CAPs recorded at the pudendal nerve after an L7-Sl anastomosis was similar for both dorsal and ventral roots. Our studies have also shown that the normal contralateral L 7 root has a limited projection to the pudendal nerve. However, the CAPs recorded at the pudendal nerve on the side of the anastomosed root were much larger, indicating significant redirection of fibers. Despite these encouraging results, CAP data have limited usefulness. Although CAPs give an indication of axonal regeneration with respect to the number of axons crossing a repair site, they cannot predict the number of proximal axons with distal connections. Similarly, no definitive evidence for functional reinnervation can be derived from CAP data. 10 Previous studies have suggested that L7 (somatic) axons can reinnervate the parasympathetically denervated bladder after nerve crossover experiments.a Recent investigations have also provided evidence that functional reinnervation of other parasympathetic ganglia by somatic axons can occur through a fraction of the synapses riormally found. 1a These observations suggest considerable redundancy in normal innervation and that return of function with somatic axons in a previously autonomically innervated structure may be possible. The mechanism of urinary bladder reinnervation by somatic axons is not known. Possible explanations include somatic axonal reinnervation of intramural ganglia and bladder smooth muscle or reinnervation of the ganglia alone as the intramural ganglia of the detrusor are merely decentralized after sacral root transection. However, temporary detrusor muscle denervation may also occur secondary to transaxonal degeneration of post-ganglionic neurons but axonal regeneration usually begins within four weeks. 14 Therefore, the process of urinary bladder reinnervation in the study outlined appears to involve somatic axonal reinnervation of intramural ganglia while the bladder smooth muscle is probably reinnervated by post ganglionic cholinergic and adrenergic axons. Urinary bladder reinnervation may also be influenced by changes in the sympathetic supply to the bladder as a consequence of parasympathetic denervation. After such denervation, a change in detrusor response can be detected at six weeks and with this change alpha adrenoreceptor function appears in the nerve terminals rather than the normal beta adrenoreceptor activity. 15 The sprouting of adrenergic nerves in the bladder and change in receptor status may be a direct result of the parasympathetic decentralization. How this local sprouting by adrenergic nerves affects subsequent reinnervation by regenerating lumbar axons is not known. In addition, sex steroids have been shown to play a role in the regulation of the density of neurotransmitter receptors and this may influence the degree of reinnervation achieved in the bladders of female individuals compared to male individuals. 16 The mechanisms of the micturition reflex are complex and controversial. However, an intact pontine micturition center appears to be important and recovery of the bladder's storage/ voiding cycle via nerve root crossover anastomoses after decentralization demands a modifiable or plastic nervous system. 17 Recently, evidence supporting the ability for synapse turnover and neuronal plasticity after injury in the mammalian nervous system was reviewed. 18· 19 Although axonal regeneration rates of one to five mm./24 hours (for sensory fibers after suture) have been documented, the time required for neuroplasticity or the development of these alternative central nervous system connections is not known. 20 Similarly, the number of appropri-
ately connected regenerated axons necessary for restoration of useful bladder function may be far fewer than anticipated because of the considerable redundance in detrusor innervation. 21 The postulate that return of bladder function after extensive neural injury is possible through collateral sprouting, sympathetic pathways or detrusor contractions initiated by intramural ganglia, appears to be no longer tenable. 8 Urinary bladder reinnervation (or reinnervation of the intramural ganglia) through nerve crossover surgery is feasible in the cat and, as observed by others, an increase in the neurologic deficit after such a technique is usually not significant clinically.2·a However, restoration of useful urinary bladder function by establishing alternative pathways for suprasacral bladder control remains to be confirmed. Presently we are performing studies examining the return of useful bladder function after crossover nerve root surgery. Other experiments using enzyme tracer techniques and investigations of somatosensory responses will also allow a better understanding of bladder function after nerve injury and reinnervation. Information from such investigations may then allow the use of similar nerve crossover techniques in selected patients with spinal cord injury or spina bifida to restore functional micturition. 22 The appropriate timing of such surgical intervention is not known yet. Furthermore, a factor contributing to the successful return of functional micturition in these patients after nerve crossover surgery will be the degree to which detrusor viability has been maintained. Detrusor fibrosis, because of chronic inflammation, could compromise return of useful bladder function because of myogenic rather than neurogenic factors.
Acknowledgments. The authors are grateful for the support and encouragement extended by Edward McGuire, M.D., Luis de Medinaceli, M.D. and William de Groat, Ph.D. REFERENCES
1. Vorstman, B., Schlossberg, S. and Kass, L.: Can severe spinal nerve injury be repaired? JAMA, 254: 55, 1985. 2. Kilvington, B.: An investigation on the regeneration of nerves with regard to surgical treatment of certain paralyses. Br. Med. J., 1: 988, 1907. 3. Sundin, T.: Reinnervation of the urinary bladder. An experimental study in cats. Scand. J. Urol. Nephrol. (Suppl.), 17: 3, 1972. 4. Carlsson, C. A. and Sundin, T.: Reconstruction of afferent and efferent nervous pathways to the urinary bladder in two paraplegic patients. Spine, 5: 37, 1980. 5. Carlsson, C. A. and Sundin, T.: Forefront: preliminary report. Reconstruction of efferent pathways to the urinary bladder in a paraplegic child. Review Surg., 24: 73, 1967. 6. Conzen, M. A. and Sollmann, H.: Reinnervation of the urinary bladder after microsurgical reconstruction of transected caudal fibers: an experimental study in pigs. Urol. Res., 10: 141, 1982. 7. McGuire, E. J. and Morrissey, S. G.: The development of reflex bladder activity following spinal cord injury in cats and a method to control it. Neurourol. Urodynam., 1: 211, 1982. 8. Carlsson, C. A. and Sundin, T.: Reconstruction of severed ventral roots innervating the urinary bladder: an experimental study in cats. Scand. J. Urol. Nephrol., 2: 199, 1968. 9. Sundin, T. and Carlsson, C. A.: Reconstruction of severed dorsal roots innervating the urinary bladder: an experimental study in cats. II. Regeneration studies. Scand. J. Urol. Nephrol., 6: 185, 1972. 10. Rosen, J.M. and Jewett, D. L.: Physiological methods of evaluating experimental nerve repairs. In: Nerve Repair and Regeneration. Its Clinical and Experimental Basis. Edited by D. L. Jewett and H. R. McCarroll. St. Louis: The C.V. Mosby Co., ch. 15, p. 150, 1979. 11. de Medinaceli, L., Wyatt, R. J. and Freed, W. J.: Peripheral nerve reconnection: mechanical, thermal and ionic conditions that promote the return of function. Exp. Neurol., 81: 469, 1983. 12. Coggeshall, R. E. and Ito, H.: Sensory fibers in ventral roots L7 and 81 in the cat. J. Physiol., 267: 215, 1977.
969 13. Proctor, Vv., Roper, S. and Taylor, B.: Somatic axons can innervate the autonomic neurones in the frog heart. J. Physiol., 326: 173, 1982. 14. Elbadawi, A., Atta, M.A. and Franck, J. I.: Intrinsic neuromuscular defects in the neurogenic bladder. I. Short-term ultrastructural changes in muscular innervation of the decentralized feline bladder base following unilateral sacral ventral rhizotomy. Neurourol. Urodynam., 3: 93, 1984. 15. Sundin, T. and Dahlstrom, A.: The sympathetic innervation of the urinary bladder and urethra in the normal state and after parasympathetic denervation at the spinal root level. Scand. J. Urol. Nephrol., 7: 131, 1973. 16. Levin, R. M., Jacobowitz, D. and Wein, A. J.: Autonomic innervation of rabbit urinary bladder following estrogen administration. Urology, 17: 449, 1981. 17. de Groat, W. C.: Nervous control of the urinary bladder of the cat. Brain Res., 87: 201, 1975.
18. Freed, W. J., de Medinaceli, L. and Wyatt, R. J.: Promoting functional plasticity in the damaged nervous system. Science, 227: 1544, 1985. 19. Cotman, C. W. and Nieto-Sampedro, M.: Cell biology of synaptic plasticity. Science, 225: 1287, 1984. 20. Sunderland, S.: Nerves and Nerve Injuries. Churchill Livingston, New York, Second Edition, p. 121, 1978. 21. Gero!, A. Y., Seymour, R. J., Pava, A. A., Robertson, J. W., Boyesen, S., Callan, D. and Campbell, J.: Evidence of reserve in the innervation of the urinary bladder of cats: a preliminary study directed at improving the therapeutic approach to bladder dysfunction in man following cauda equina injury. Surg. Forum, 6: 499, 1955. 22. Vorstman, B., Schlossberg, S., Kass, L., Devine, C. J. Jr. and Horton, C. E.: Spinal nerve root surgery for urinary bladder reinnervation. Neurourol. Urodynam., 5: 327, 1986.