Journal of the Autonomic Nervous System, 41 (1992) 197-208
197
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01333
Hypogastric nerve section reveals a role for both afferent and efferent fibres in the feline continence process C h r i s t o p h e r W. V a u g h a n Gordon Craig Laboratory, Department of Surgery, University of Sydney, Sydney, Australia (Received 14 May 1992) (Revision received and accepted 28 July 1992)
Key words: Continence; Hypogastric nerve; Bladder; Physiological filling; Cat Abstract The role of the hypogastric afferent and efferent innervation in the process of urine storage during natural rate filling was examined in the pentobarbitone anaesthetized cat. The observed changes in bladder pressure following hypogastric nerve section demonstrated the presence of baseline sympatho-inhibitory and phasic sympatho-excitatory influences for a significant portion of each distension, which proceeded to a volume just over halfway through the continence process. A reduction in bladder wall compliance at the end of the distensions provided evidence for a net sympatho-inhibitory influence in the second half of the continence phase. Hypogastric nerve section also resulted in a reduction in the reflex increase in hypogastric efferent nerve activity, measured in-continuity. This suggested that hypogastric afferent fibres made a significant contribution to the regulation of sympathetic hypogastric nerve activity during natural rate filling.
Introduction
The role of the extrinsic innervation of the bladder in the process of urine storage throughout the continence, or filling phase is controversial [19]. While there is evidence to support the proposition that hypogastric efferent nerve activity has a net inhibitory action on the feline detrusor muscle during rapid filling [7,10], there is some doubt over its role during filling at naturally occurring rates [16]. Thus it is widely believed that the accommodative ability of the bladder during the continence phase is largely due to the
Correspondence to: C.W. Vaughan, Gordon Craig Laboratory, Department of Surgery, University of Sydney, Sydney, N.S.W. 2006, Australia.
mechanical properties of the bladder wall [17,22,27]. In contrast, we have recently demonstrated sympatho-inhibitory and sympatho-excitatory influences over the feline detrusor muscle at a volume just over halfway through the continence phase [29]. It has yet to be determined how the sympathetic influences previously observed at constant volume might be involved in the regulation of the bladder detrusor during natural rate filling. In the present study the role of the sympathetic hypogastric efferent outflow in the continence process was examined by measuring the effect of hypogastric nerve section on the natural rate cystometrogram. Because nerve section interrupted afferent as well as efferent fibres, the simultaneous measurement of hypogastric nerve activity afforded an opportunity to re-examine
198
the disputed role of hypogastric afferent fibres in the regulation of hypogastric efferent nerve activity.
Materials and Methods Experiments were carried out in six male cats (4.2-6.3 kg). Food was withdrawn for 24-36 h, there being a free supply of water. An hour before the anaesthetic induction, an enema (Microlax microenema, 5 ml) was given. Anaesthesia was commenced with intramuscular ketamine hydrochloride (Ketalar, 15 mg kg -1) and followed by intravenous sodium pentobarbitone (Nembutal, 30 mg kg l). A tracheostomy was performed and a tracheal tube inserted. Femoral arterial pressure was monitored (Sanborn 267B). An anaesthetic and fluid infusion (0.72 mg ml - I in 4% dextrose and 0.18% saline at 4 ml kg -~ min 1) was given via a femoral vein catheter (McGaw Volumetric Infusion Pump). Anaesthetic level was monitored in spontaneously breathing animals by clinical signs and the carbon dioxide content of expired air (Datex CD102). The anaesthetic infusion rate was adjusted to keep the expired percentage of carbon dioxide as constant as possible, the mean variation in each animal being 0.4% (range 0.3-0.7%). The mean value for all the recordings in all animals was 4.3% (range 4.04.8%). The acid-base status, PaCO2 and haematocrit were measured (Nova Stat profile 4) and maintained within normal limits. Oesophageal temperature was maintained at 37.6-38.1°C by a heating pad. Bladder isolation and ner~;e preparation In supine animals, the bladder was exposed by a lower abdominal midline incision. A ventral slit was made in the urethra, a twin barrel stainless steel catheter inserted and the bladder drained. The outer barrel of the catheter was connected to a syringe p u m p (Braun 1833). The central barrel was used to measure bladder pressure (Sanborn 268B). In isolating the bladder, both ureters were tied 3-5 cm from the bladder wall and the proximal ends cannulated to monitor the renal output.
In order to facilitate unhindered bladder expansion, the colon was positioned on the left side of the abdominal cavity and the small intestine retracted cranially. The bladder and the abdominal contents were covered by paraffin oil and maintained between 37.0-38.5°C by an overhead lamp. The pressure of the paraffin pool was measured via a wide bore catheter (Sanborn 268B). The left hypogastric nerve was cleared of the surrounding fascia for a distance of approximately 25 mm and placed over silver silver-chloride electrodes for in-continuity recording (interelectrode distance 10mm). The nerve blood supply was interfered with as little as possible and nerve direction changes were kept to a minimum. In-continuity nerve activity was filtered at 5-1 000 Hz. The fascial attachments of a small segment of nerve were cleared 20-30 mm distally to the recording site. A similar space was created around the contralateral nerve, care being taken to ensure that the nerves were not damaged. Local anaesthetic (1% xylocaine) could be injected into the space around each nerve and the nerve then crushed using fine forceps. Care was taken that the nerve was not pulled, that the electrodes remained untouched and that the spread of local anaesthetic was minimal. Procedures The normal rate of feline urine output is 1.1 ml kg ~ h -1 and can be produced naturally at up to fifteen times this rate [16]. In order that the continence periods were not extremely prolonged, the natural filling rate used in the present study was 30 or 45 ml h - 1, which corresponded to a rate of 8.8 (range 7.1-10.7) ml kg i h-1. The infusate was a urine substitute [25,30]. Distensions were terminated at twice the volume at which hypogastric nerve activity began to increase. A previous study has indicated that this filling volume is 62% (range 42-90%) of the volume at which micturition, characterised by the onset of parasympathetic nerve activity, occurs in the pentobarbitone anaesthetized cat [25]. Thus, the filling volumes achieved in the present study, of 9.6 (range 6.3-11.8) ml kg -~, were likely to be just over halfway through the continence phase. It might be noted that the volume at which mic-
199 turition occurs in the conscious cat has been reported as being 28.0 (range 10.2-64.8) ml kg-1 [30]. The experimental protocol consisted of three successive distensions, the bladder being allowed to drain freely for a period of 20 min between each distension. The first distension was not used for analysis because of 'non-specific' and 'firstpass' effects on nerve activity and bladder wall motility [28]. In order to determine the effects of hypogastric nerve section, both hypogastric nerves were crushed at the end of the second distension (see above).
Analysis In-continuity hypogastric nerve activity, bladder pressure and the pressure in the paraffin pool were recorded on magnetic tape (Racal Store 14DS). The analyses were carried out with an IBM-AT microcomputer running ASYST. Bladder pressure and paraffin pool pressure recordings were low-pass filtered (2 Hz) and digitised (bandwidth DC-0.5 Hz, sampling rate 2 Hz). Measured bladder pressure reflected the transmural pressure due to bladder wall tension plus the hydrostatic pressure of the fluid above the manometer head, both inside and outside the bladder [16]. Measured paraffin pool pressure reflected the hydrostatic pressure above the manometer head. Thus, transmural pressure was calculated by subtracting the measured paraffin pool pressure from the measured bladder pressure, with both manometer heads at the same height. Bladder wall tension, or more appropriately strain T (gram-force cm -2) was calculated at the inner surface of the bladder wall using the formula for a thick spherical shell [28]. Hypogastric nerve activity was high-pass filtered (5 Hz), full-wave rectified and integrated over each sample period (/~V s (0.5 s)-l). The noise contribution was determined by measuring the mean integrated activity over 2 min after local anaesthetic (1% xylocaine) had been placed on the nerve. This value was subtracted from the sampled integrated nerve activity. Measures of the mean transmural bladder pressure, bladder wall tension and integrated incontinuity hypogastric nerve activity were calcu-
lated over successive 0.5 ml k g - t volume increments for each distension. The feline cystometrogram has been described as comprising a gradual increase in baseline pressure and the variable appearance of phasic non-micturating contractions (NMCs) [17]. Independent analysis of these two components of the mean pressure level was carried out because of differences in their underlying mechanisms [29]. A baseline segment was defined as the phase between consecutive NMCs which did not exceed the minimum baseline pressure for that segment by more than 1 cm H 2 0 [29]. Baseline pressure, tension and nerve activity were calculated as the average level over each baseline segment. In the absence of NMCs greater than 1 cm H 2 0 , consecutive 0.5 ml kg -1 epochs were used for the baseline analysis. NMC amplitude was calculated as the difference between the peak contraction pressure and the minimum pressure during the preceding baseline segment. The effect of hypogastric nerve section on the cystometrogram was assessed by comparing the increase in mean and baseline pressure over a given volume increment for the two distensions. This represented a simple measure of bladder wall compliance. The effect of hypogastric nerve section on the reflex increase in mean and baseline hypogastric 'efferent' nerve activity during bladder filling was examined by recording in-continuity nerve activity, proximally to the site of nerve section. Integrated efferent nerve activity was plotted against derived bladder wall tension for the two distensions. In a similar preparation, it has been shown that the tension-efferent nerve activity relationship is reproducible during successive distensions and independent of any changes in compliance [28]. Tension thresholds were measured by identifying two successive points at which hypogastric nerve activity had increased by one standard deviation above the mean resting level. The level of reflex activation was measured as the increase in hypogastric nerve activity above the resting level at twice the pressure, or tension threshold level. Statistical comparisons between the second (hypogastric nerves intact) and third distensions (hypogastric nerves crushed) were made using Wilcoxon's signed-rank test for paired data, a
2OO
P-value of less than 0.05 indicating a significant difference [26].
Results
Effect of hypogastric nerue section on the cystometrogram When both hypogastric nerves were intact, the cystometrogram was characterised by a gradual increase in the level of baseline pressure and the emergence of small irregular contractions, usually less than 1 cm H 2 0 in amplitude (Fig. la) [24]. At some point in the filling cycle (4.6, range 1.7-6.5 ml kg-~), larger NMCs appeared (5.1, range 2.2-8.2 cm H 2 0 ) which became more regular and decreased in amplitude as filling progressed [27]. The most obvious effect of bilateral hypogastric nerve section was a decrease in the number and amplitude of NMCs (Fig. lb), there being a complete abolition of NMCs in two animals. Following nerve section, NMCs greater than 1 cm H 2 0 were significantly reduced in number
(14, range 0 - 3 8 % of the intact number) and amplitude (3.7, range 1.2-6.9 cm H 2 0 reduction in mean amplitude) ( P < 0.05). While nerve section had no effect on the increase in mean or baseline pressure at the point at which hypogastric nerve activity began to increase ( P > 0.05), changes in compliance became more evident as filling progressed (Figs. 2,3). The increase in mean transmural pressure over each distension when both nerves were intact (3.1, range 2.3-4.5 cm H 2 0 ) was similar to that at a corresponding volume after nerve section (3.2, range 1.9-4.4 cm H 2 0 ) ( P > 0.05; Fig. 2). However, the increase in mean pressure over the last 1 ml kg-~ was greater after nerve section (0.6, range 0.3-1.1 cm H 2 0 ) than when both nerves were intact (0.2, range 0.1-0.5 cm H 2 0 ) ( P < 0.05). Analysis of baseline pressure changes, which excluded N M C effects, revealed more dramatic differences. When both nerves were intact, there was often a 'linear' increase in baseline pressure throughout each distension (Fig. 3d-f), while in
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Integrated HGN Activity
/~.V.s 0.5s
lOmin Fig. l. Typical response of transmural bladder pressure and integrated hypogastric in-continuity nerve (HGN) activity during a slow distension, when both hypogastric nerves were intact (A) and after bilateral hypogastric nerve crushing (B). Nerve activity was integrated (~zV s (0.5 s) ~) and smoothed. The first distension was preceded by a similar slow distension. The first NMC (contraction > l cm H e 0 ) in each distension is indicated by an arrow.
201
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Blodder Filling Volume (ml.kg -1) Fig. 2. Relationship between mean transmural bladder pressure and bladder filling volume during a slow distension when both hypogastric nerves were intact (closed circle) and after bilateral hypogastricnerve crushing (open circle), in each of the six animals (a-f). Each point is the mean for a 0.5 ml kg-x volume increment. A slow distension was carried out in each animal prior to the two distensions shown here.
other animals, there was a distinct reduction in the increase in baseline pressure above the volume at which hypogastric nerve activity began to increase (Fig. 3a-c). Nerve section usually resuited in an exponential-like increase in baseline pressure towards the end of each distension (Fig. 3). In all animals, the increase in baseline transmural pressure over each distension was greater after nerve section (2.9, range 2.1-3.5 cm H 2 0 ) , than at a corresponding volume when both hypogastric nerves were intact (2.3, range 2.0-2.6 cm H 2 0 ) ( P < 0.05). Similarly, the increase in baseline pressure over the last 1 ml kg -1 was greater after nerve section (0.7, range 0.3-1.3 cm H 2 0 ) than when both nerves were intact (0.2, range 0.1-0.3 cm H 2 0 ) ( P < 0.05).
Effect of hypogastric nerve section on the hypogastric reflex response The mean level of in-continuity hypogastric 'efferent' nerve activity, with the bladder empty, was not affected by nerve section, being 0.30 (range 0.08-0.62) ~V s (0.5 s) -1 at the start of the second distension and 0.32 (range 0.08-0.63) /t/,g S (0.5 S)-1 at the start of the third distension. The reflex increase in mean hypogastric nerve activity appeared to be reduced by nerve section in four out of six animals (Fig. 4b, d-f), a change being less obvious in the other two animals (Fig. 4a,c). In all animals, the mean tension threshold for an increase in hypogastric nerve activity was greater after nerve section (17, range 6 - 2 2 gF cm -1) than when both nerves were intact (10,
202 r a n g e 4 - 1 7 g F cm - 2 ) ( P < 0.05). T h e m e a n level of h y p o g a s t r i c nerve activity at twice the tension t h r e s h o l d was g r e a t e r b e f o r e nerve section (0.70, r a n g e 0.42-1.19 tzV s (0.5 s ) - l ) , t h a n at a corres p o n d i n g level of t e n s i o n w h e n b o t h nerves were intact (0.52, r a n g e 0 . 2 1 - 0 . 9 8 / x V s (0.5 s ) - ~) ( P < 0.05). Thus, the reflex i n c r e a s e in m e a n hypogastric nerve activity at twice t h e tension t h r e s h o l d was r e d u c e d by 47% ( r a n g e 1 3 - 6 7 % ) a f t e r nerve section. N e r v e section h a d a similar effect on the inc r e a s e in b a s e l i n e h y p o g a s t r i c nerve activity (Fig. 5). In all animals, the b a s e l i n e t e n s i o n t h r e s h o l d for an i n c r e a s e in h y p o g a s t r i c nerve activity was g r e a t e r after nerve section (19, r a n g e 6 - 2 6 g F c m - 2 ) , t h a n w h e n b o t h nerves were intact (12, r a n g e 3 - 2 1 g F cm - 2 ) ( P < 0.05). T h e b a s e l i n e
level of h y p o g a s t r i c nerve activity at twice thc tension t h r e s h o l d was g r e a t e r b e f o r e nerve section (0.89, r a n g e 0,42-1.63 tzV s (0.5 s) t), than at a c o r r e s p o n d i n g level of tension when both nerves w e r e intact (0.56, range 0.18-1.07 # V s (0.5 s) J) ( P < 0.05). Thus, the reflex increase in b a s e l i n e hypogastric nerve activity at twice the tension t h r e s h o l d was r e d u c e d by 61% (range 2 2 - 8 3 % ) after nerve section.
Contribution o f a f f e r e n t / e f f e r e n t actiuity to the in-continuity hypogastric nerce recording I n - c o n t i n u i t y hypogastric nerve activity is the sum o f the p o t e n t i a l s from pre- a n d p o s t g a n glionic fibres, a n d from afferent fibres. In two animals, the relative c o n t r i b u t i o n of a f f e r e n t and e f f e r e n t nerve fibre p o t e n t i a l s to i n t e g r a t e d in-
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Blodder FTI]incj Volume (ml.k9 -1) Fig. 3. Relationship between baseline transmural bladder pressure and bladder filling volume during a slow distension when both hypogastric nerves were intact (closed circle) and after bilateral hypogastric nerve crush (open circle). The baseline segments were measured between consecutive NMCs (see Materials and Methods). The arrows indicate the point at which in-continuity hypogastric nerve activity began to increase during the hypogastric nerve intact distension (closed circle). The data for different experiments displayed in Figs. 2 and 3 are laid out in an identical manner.
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197
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01333
Hypogastric nerve section reveals a role for both afferent and efferent fibres in the feline continence process C h r i s t o p h e r W. V a u g h a n Gordon Craig Laboratory, Department of Surgery, University of Sydney, Sydney, Australia (Received 14 May 1992) (Revision received and accepted 28 July 1992)
Key words: Continence; Hypogastric nerve; Bladder; Physiological filling; Cat Abstract The role of the hypogastric afferent and efferent innervation in the process of urine storage during natural rate filling was examined in the pentobarbitone anaesthetized cat. The observed changes in bladder pressure following hypogastric nerve section demonstrated the presence of baseline sympatho-inhibitory and phasic sympatho-excitatory influences for a significant portion of each distension, which proceeded to a volume just over halfway through the continence process. A reduction in bladder wall compliance at the end of the distensions provided evidence for a net sympatho-inhibitory influence in the second half of the continence phase. Hypogastric nerve section also resulted in a reduction in the reflex increase in hypogastric efferent nerve activity, measured in-continuity. This suggested that hypogastric afferent fibres made a significant contribution to the regulation of sympathetic hypogastric nerve activity during natural rate filling.
Introduction
The role of the extrinsic innervation of the bladder in the process of urine storage throughout the continence, or filling phase is controversial [19]. While there is evidence to support the proposition that hypogastric efferent nerve activity has a net inhibitory action on the feline detrusor muscle during rapid filling [7,10], there is some doubt over its role during filling at naturally occurring rates [16]. Thus it is widely believed that the accommodative ability of the bladder during the continence phase is largely due to the
Correspondence to: C.W. Vaughan, Gordon Craig Laboratory, Department of Surgery, University of Sydney, Sydney, N.S.W. 2006, Australia.
mechanical properties of the bladder wall [17,22,27]. In contrast, we have recently demonstrated sympatho-inhibitory and sympatho-excitatory influences over the feline detrusor muscle at a volume just over halfway through the continence phase [29]. It has yet to be determined how the sympathetic influences previously observed at constant volume might be involved in the regulation of the bladder detrusor during natural rate filling. In the present study the role of the sympathetic hypogastric efferent outflow in the continence process was examined by measuring the effect of hypogastric nerve section on the natural rate cystometrogram. Because nerve section interrupted afferent as well as efferent fibres, the simultaneous measurement of hypogastric nerve activity afforded an opportunity to re-examine
198
the disputed role of hypogastric afferent fibres in the regulation of hypogastric efferent nerve activity.
Materials and Methods Experiments were carried out in six male cats (4.2-6.3 kg). Food was withdrawn for 24-36 h, there being a free supply of water. An hour before the anaesthetic induction, an enema (Microlax microenema, 5 ml) was given. Anaesthesia was commenced with intramuscular ketamine hydrochloride (Ketalar, 15 mg kg -1) and followed by intravenous sodium pentobarbitone (Nembutal, 30 mg kg l). A tracheostomy was performed and a tracheal tube inserted. Femoral arterial pressure was monitored (Sanborn 267B). An anaesthetic and fluid infusion (0.72 mg ml - I in 4% dextrose and 0.18% saline at 4 ml kg -~ min 1) was given via a femoral vein catheter (McGaw Volumetric Infusion Pump). Anaesthetic level was monitored in spontaneously breathing animals by clinical signs and the carbon dioxide content of expired air (Datex CD102). The anaesthetic infusion rate was adjusted to keep the expired percentage of carbon dioxide as constant as possible, the mean variation in each animal being 0.4% (range 0.3-0.7%). The mean value for all the recordings in all animals was 4.3% (range 4.04.8%). The acid-base status, PaCO2 and haematocrit were measured (Nova Stat profile 4) and maintained within normal limits. Oesophageal temperature was maintained at 37.6-38.1°C by a heating pad. Bladder isolation and ner~;e preparation In supine animals, the bladder was exposed by a lower abdominal midline incision. A ventral slit was made in the urethra, a twin barrel stainless steel catheter inserted and the bladder drained. The outer barrel of the catheter was connected to a syringe p u m p (Braun 1833). The central barrel was used to measure bladder pressure (Sanborn 268B). In isolating the bladder, both ureters were tied 3-5 cm from the bladder wall and the proximal ends cannulated to monitor the renal output.
In order to facilitate unhindered bladder expansion, the colon was positioned on the left side of the abdominal cavity and the small intestine retracted cranially. The bladder and the abdominal contents were covered by paraffin oil and maintained between 37.0-38.5°C by an overhead lamp. The pressure of the paraffin pool was measured via a wide bore catheter (Sanborn 268B). The left hypogastric nerve was cleared of the surrounding fascia for a distance of approximately 25 mm and placed over silver silver-chloride electrodes for in-continuity recording (interelectrode distance 10mm). The nerve blood supply was interfered with as little as possible and nerve direction changes were kept to a minimum. In-continuity nerve activity was filtered at 5-1 000 Hz. The fascial attachments of a small segment of nerve were cleared 20-30 mm distally to the recording site. A similar space was created around the contralateral nerve, care being taken to ensure that the nerves were not damaged. Local anaesthetic (1% xylocaine) could be injected into the space around each nerve and the nerve then crushed using fine forceps. Care was taken that the nerve was not pulled, that the electrodes remained untouched and that the spread of local anaesthetic was minimal. Procedures The normal rate of feline urine output is 1.1 ml kg ~ h -1 and can be produced naturally at up to fifteen times this rate [16]. In order that the continence periods were not extremely prolonged, the natural filling rate used in the present study was 30 or 45 ml h - 1, which corresponded to a rate of 8.8 (range 7.1-10.7) ml kg i h-1. The infusate was a urine substitute [25,30]. Distensions were terminated at twice the volume at which hypogastric nerve activity began to increase. A previous study has indicated that this filling volume is 62% (range 42-90%) of the volume at which micturition, characterised by the onset of parasympathetic nerve activity, occurs in the pentobarbitone anaesthetized cat [25]. Thus, the filling volumes achieved in the present study, of 9.6 (range 6.3-11.8) ml kg -~, were likely to be just over halfway through the continence phase. It might be noted that the volume at which mic-
199 turition occurs in the conscious cat has been reported as being 28.0 (range 10.2-64.8) ml kg-1 [30]. The experimental protocol consisted of three successive distensions, the bladder being allowed to drain freely for a period of 20 min between each distension. The first distension was not used for analysis because of 'non-specific' and 'firstpass' effects on nerve activity and bladder wall motility [28]. In order to determine the effects of hypogastric nerve section, both hypogastric nerves were crushed at the end of the second distension (see above).
Analysis In-continuity hypogastric nerve activity, bladder pressure and the pressure in the paraffin pool were recorded on magnetic tape (Racal Store 14DS). The analyses were carried out with an IBM-AT microcomputer running ASYST. Bladder pressure and paraffin pool pressure recordings were low-pass filtered (2 Hz) and digitised (bandwidth DC-0.5 Hz, sampling rate 2 Hz). Measured bladder pressure reflected the transmural pressure due to bladder wall tension plus the hydrostatic pressure of the fluid above the manometer head, both inside and outside the bladder [16]. Measured paraffin pool pressure reflected the hydrostatic pressure above the manometer head. Thus, transmural pressure was calculated by subtracting the measured paraffin pool pressure from the measured bladder pressure, with both manometer heads at the same height. Bladder wall tension, or more appropriately strain T (gram-force cm -2) was calculated at the inner surface of the bladder wall using the formula for a thick spherical shell [28]. Hypogastric nerve activity was high-pass filtered (5 Hz), full-wave rectified and integrated over each sample period (/~V s (0.5 s)-l). The noise contribution was determined by measuring the mean integrated activity over 2 min after local anaesthetic (1% xylocaine) had been placed on the nerve. This value was subtracted from the sampled integrated nerve activity. Measures of the mean transmural bladder pressure, bladder wall tension and integrated incontinuity hypogastric nerve activity were calcu-
lated over successive 0.5 ml k g - t volume increments for each distension. The feline cystometrogram has been described as comprising a gradual increase in baseline pressure and the variable appearance of phasic non-micturating contractions (NMCs) [17]. Independent analysis of these two components of the mean pressure level was carried out because of differences in their underlying mechanisms [29]. A baseline segment was defined as the phase between consecutive NMCs which did not exceed the minimum baseline pressure for that segment by more than 1 cm H 2 0 [29]. Baseline pressure, tension and nerve activity were calculated as the average level over each baseline segment. In the absence of NMCs greater than 1 cm H 2 0 , consecutive 0.5 ml kg -1 epochs were used for the baseline analysis. NMC amplitude was calculated as the difference between the peak contraction pressure and the minimum pressure during the preceding baseline segment. The effect of hypogastric nerve section on the cystometrogram was assessed by comparing the increase in mean and baseline pressure over a given volume increment for the two distensions. This represented a simple measure of bladder wall compliance. The effect of hypogastric nerve section on the reflex increase in mean and baseline hypogastric 'efferent' nerve activity during bladder filling was examined by recording in-continuity nerve activity, proximally to the site of nerve section. Integrated efferent nerve activity was plotted against derived bladder wall tension for the two distensions. In a similar preparation, it has been shown that the tension-efferent nerve activity relationship is reproducible during successive distensions and independent of any changes in compliance [28]. Tension thresholds were measured by identifying two successive points at which hypogastric nerve activity had increased by one standard deviation above the mean resting level. The level of reflex activation was measured as the increase in hypogastric nerve activity above the resting level at twice the pressure, or tension threshold level. Statistical comparisons between the second (hypogastric nerves intact) and third distensions (hypogastric nerves crushed) were made using Wilcoxon's signed-rank test for paired data, a
2OO
P-value of less than 0.05 indicating a significant difference [26].
Results
Effect of hypogastric nerue section on the cystometrogram When both hypogastric nerves were intact, the cystometrogram was characterised by a gradual increase in the level of baseline pressure and the emergence of small irregular contractions, usually less than 1 cm H 2 0 in amplitude (Fig. la) [24]. At some point in the filling cycle (4.6, range 1.7-6.5 ml kg-~), larger NMCs appeared (5.1, range 2.2-8.2 cm H 2 0 ) which became more regular and decreased in amplitude as filling progressed [27]. The most obvious effect of bilateral hypogastric nerve section was a decrease in the number and amplitude of NMCs (Fig. lb), there being a complete abolition of NMCs in two animals. Following nerve section, NMCs greater than 1 cm H 2 0 were significantly reduced in number
(14, range 0 - 3 8 % of the intact number) and amplitude (3.7, range 1.2-6.9 cm H 2 0 reduction in mean amplitude) ( P < 0.05). While nerve section had no effect on the increase in mean or baseline pressure at the point at which hypogastric nerve activity began to increase ( P > 0.05), changes in compliance became more evident as filling progressed (Figs. 2,3). The increase in mean transmural pressure over each distension when both nerves were intact (3.1, range 2.3-4.5 cm H 2 0 ) was similar to that at a corresponding volume after nerve section (3.2, range 1.9-4.4 cm H 2 0 ) ( P > 0.05; Fig. 2). However, the increase in mean pressure over the last 1 ml kg-~ was greater after nerve section (0.6, range 0.3-1.1 cm H 2 0 ) than when both nerves were intact (0.2, range 0.1-0.5 cm H 2 0 ) ( P < 0.05). Analysis of baseline pressure changes, which excluded N M C effects, revealed more dramatic differences. When both nerves were intact, there was often a 'linear' increase in baseline pressure throughout each distension (Fig. 3d-f), while in
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Integrated HGN Activity
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lOmin Fig. l. Typical response of transmural bladder pressure and integrated hypogastric in-continuity nerve (HGN) activity during a slow distension, when both hypogastric nerves were intact (A) and after bilateral hypogastric nerve crushing (B). Nerve activity was integrated (~zV s (0.5 s) ~) and smoothed. The first distension was preceded by a similar slow distension. The first NMC (contraction > l cm H e 0 ) in each distension is indicated by an arrow.
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Blodder Filling Volume (ml.kg -1) Fig. 2. Relationship between mean transmural bladder pressure and bladder filling volume during a slow distension when both hypogastric nerves were intact (closed circle) and after bilateral hypogastricnerve crushing (open circle), in each of the six animals (a-f). Each point is the mean for a 0.5 ml kg-x volume increment. A slow distension was carried out in each animal prior to the two distensions shown here.
other animals, there was a distinct reduction in the increase in baseline pressure above the volume at which hypogastric nerve activity began to increase (Fig. 3a-c). Nerve section usually resuited in an exponential-like increase in baseline pressure towards the end of each distension (Fig. 3). In all animals, the increase in baseline transmural pressure over each distension was greater after nerve section (2.9, range 2.1-3.5 cm H 2 0 ) , than at a corresponding volume when both hypogastric nerves were intact (2.3, range 2.0-2.6 cm H 2 0 ) ( P < 0.05). Similarly, the increase in baseline pressure over the last 1 ml kg -1 was greater after nerve section (0.7, range 0.3-1.3 cm H 2 0 ) than when both nerves were intact (0.2, range 0.1-0.3 cm H 2 0 ) ( P < 0.05).
Effect of hypogastric nerve section on the hypogastric reflex response The mean level of in-continuity hypogastric 'efferent' nerve activity, with the bladder empty, was not affected by nerve section, being 0.30 (range 0.08-0.62) ~V s (0.5 s) -1 at the start of the second distension and 0.32 (range 0.08-0.63) /t/,g S (0.5 S)-1 at the start of the third distension. The reflex increase in mean hypogastric nerve activity appeared to be reduced by nerve section in four out of six animals (Fig. 4b, d-f), a change being less obvious in the other two animals (Fig. 4a,c). In all animals, the mean tension threshold for an increase in hypogastric nerve activity was greater after nerve section (17, range 6 - 2 2 gF cm -1) than when both nerves were intact (10,
202 r a n g e 4 - 1 7 g F cm - 2 ) ( P < 0.05). T h e m e a n level of h y p o g a s t r i c nerve activity at twice the tension t h r e s h o l d was g r e a t e r b e f o r e nerve section (0.70, r a n g e 0.42-1.19 tzV s (0.5 s ) - l ) , t h a n at a corres p o n d i n g level of t e n s i o n w h e n b o t h nerves were intact (0.52, r a n g e 0 . 2 1 - 0 . 9 8 / x V s (0.5 s ) - ~) ( P < 0.05). Thus, the reflex i n c r e a s e in m e a n hypogastric nerve activity at twice t h e tension t h r e s h o l d was r e d u c e d by 47% ( r a n g e 1 3 - 6 7 % ) a f t e r nerve section. N e r v e section h a d a similar effect on the inc r e a s e in b a s e l i n e h y p o g a s t r i c nerve activity (Fig. 5). In all animals, the b a s e l i n e t e n s i o n t h r e s h o l d for an i n c r e a s e in h y p o g a s t r i c nerve activity was g r e a t e r after nerve section (19, r a n g e 6 - 2 6 g F c m - 2 ) , t h a n w h e n b o t h nerves were intact (12, r a n g e 3 - 2 1 g F cm - 2 ) ( P < 0.05). T h e b a s e l i n e
level of h y p o g a s t r i c nerve activity at twice thc tension t h r e s h o l d was g r e a t e r b e f o r e nerve section (0.89, r a n g e 0,42-1.63 tzV s (0.5 s) t), than at a c o r r e s p o n d i n g level of tension when both nerves w e r e intact (0.56, range 0.18-1.07 # V s (0.5 s) J) ( P < 0.05). Thus, the reflex increase in b a s e l i n e hypogastric nerve activity at twice the tension t h r e s h o l d was r e d u c e d by 61% (range 2 2 - 8 3 % ) after nerve section.
Contribution o f a f f e r e n t / e f f e r e n t actiuity to the in-continuity hypogastric nerce recording I n - c o n t i n u i t y hypogastric nerve activity is the sum o f the p o t e n t i a l s from pre- a n d p o s t g a n glionic fibres, a n d from afferent fibres. In two animals, the relative c o n t r i b u t i o n of a f f e r e n t and e f f e r e n t nerve fibre p o t e n t i a l s to i n t e g r a t e d in-
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Blodder FTI]incj Volume (ml.k9 -1) Fig. 3. Relationship between baseline transmural bladder pressure and bladder filling volume during a slow distension when both hypogastric nerves were intact (closed circle) and after bilateral hypogastric nerve crush (open circle). The baseline segments were measured between consecutive NMCs (see Materials and Methods). The arrows indicate the point at which in-continuity hypogastric nerve activity began to increase during the hypogastric nerve intact distension (closed circle). The data for different experiments displayed in Figs. 2 and 3 are laid out in an identical manner.
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