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RESEARCH |
1 Departamento de Fisiología, Biofísica y Neurociencias2 Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados CINVESTAV del IPN, Apartado Postal 14-740, 07360 México D. F., México
Correspondence should be addressed to E J Muñoz-Martínez; Email: jmunoz{at}fisio.cinvestav.mx
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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The present paper shows the innervation, the mechanics and the motor response to vestibule penetration as well as the structure of a sphincter in the wall of the vaginal vestibule. Concerning the innervation, it is a dogma that each fibre in striated muscles receives a single endplate, but several facts show that some fibres might receive more than one endplate from different axons (Jarcho et al. 1952, Rossi 1990, Zenker et al. 1990, Duxson & Sheard 1995, Happak et al. 1997, Perie et al. 1997, Lateva et al. 2002). Evidence of multiple innervation of vestibular constrictor sphincter (VCS) muscle fibres by both ipsilateral and contralateral MPN axons is shown here.
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Results |
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3 V, 50 µs) induced maximal Pv. L, R and LR refers in the text and figures to the left, the right or both e-MPNs or to the Pv induced by these nerves. We will call VCS the muscular apparatus producing Pv. The average peak of Pv–LR (30. 6±9.6 cm of water; N=14) was about twice the peak of unilateral Pv (Fig. 1A and B). Thus, VCS is functionally composed of halves. Average Pv peak varied among cats; the difference was not related to body weight or vestibule length (16–21 mm). Pv-R was on average larger than Pv-L (Fig. 1B), but the difference was not statistically significant.
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A bipolar stimulating electrode was fixed to the peripheral stump of e-MPN-L. Then, the cat (N=3) was placed supine in order to search ventrally for the nerve and muscle that might be involved in vestibular constriction. After removal of superficial tissues, the ischium ramus and the obturator muscles, the distal vagina appeared to be covered ventrally and laterally by the halves of the levator ani muscle (LAM; Crouch & Lackey 1969, Martin et al. 1974). LAM did not respond to e-MPN-L shock but the thin ischiocavernosus muscle (ICM; see Crouch & Lackey 1969) and the vestibular wall did. After detaching and retracting LAM and ICM, the peak Pv-L was larger (compare Pvs 1 and 2 in Fig. 1C). Caudal to LAM, fragile venous sinuses and fat covered the ventral side of the vestibule. The fat was removed and a bipolar electrode was placed over the sinuses. Single shocks induced Pv and visible twitch of the vestibule wall (se Pv 3 in Fig. 1C). No other muscles were seen to twitch.
In each cat, the timing of unilateral and bilateral Pvs was comparable. The area under Pvs was automatically computed from digital recordings using a software program (Origin 5.0, Origin Lab Corporation, Northampton, MA, USA) that can add point-to-point the area of two or more Pvs (LS); Fig. 1A and B show examples of LS. The LS of unilateral Pvs was significantly larger than Pv–LR (N=14; P=0.025, t-test for paired samples; Fig. 2) although in 3/14 cats LS and Pv–LR were approximately equal (difference <0.5%; Fig. 1B) and in one cat the latter was larger. The significant difference suggests that some VCS fibres might receive input from at least two motor axons, one from each side of the body.
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Peak Pv-L is about twice in Fig. 4 than in Fig. 2 (P=9.1 E–6). The data in Fig. 2 were obtained during late autumn and winter, and those in Fig. 4 during spring–summer and early autumn. The difference might be related to hormonal changes (see Komisaruk & Adler 1972).
Reflex e-MPN response to stimulation of the sensory pudendal nerve and to vestibule probing
With the nerves intact, SPN–L was stimulated and the electroneurogram of e-MPN-L was recorded. Single shocks induced a reflex e-MPN-L discharge with latency of 8±1.24 ms (N=14; Fig. 5A) as well as a Pv of about half the peak amplitude of the PV induced by e-MPN-L stimulation. In addition, the vestibule was probed (N=8) at 4–10 mm from the introitus with probe displacement <1 mm. Single probing with 8–12 ms long displacement of the probe induced two e-MPN-L bursts (Fig. 5B). The latency of the first burst was 1 ms from the onset of probing; at the time of the burst onset, the displacement of the probe was 60–80 µm (Fig. 5B). Given the short latency, this burst might originate in muscle afferents. The delay of the second e-MPN-L burst is about the same as that of the reflex elicited by SPN-L shock.
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The reflex induced by pudendal afferents in cats under barbiturate anaesthesia might suggest a high synaptic efficacy, resulting at least in part from reduced pre-synaptic inhibition (Weakly 1969, Nicoll 1975).
Structural findings
After fixation, the distal vagina was sectioned transversally in 5 mm blocks using scalpel (N=3). The exposed faces of each block were photographed. The caudal face in Fig. 6A is at 10 mm from the vaginal opening. At this location, the lumen is much wider than at 15 mm. In blocks fixed with glutaraldehyde–osmium, a ring of dark tissue follows the transversal contour of the wall. The ring is made of striated muscle (Fig. 6C and D). This is the vestibule constrictor sphincter (VCS) that is situated between an internal layer of smooth muscle and connective tissue and an external adventitia and venous sinuses. VCS fibres ending freely were found at the dorsal and ventral midlines (Fig. 6E). To find this was a difficult task because most muscle fibres do not end exactly at the same level and optical plane. Figure 6E shows the end of four fibres in the dorsal midline. The fibre trajectory from one end to the other and does not follow the same plane as the plane of the transversal sections of VCS. Thus, we were unable to find both ends single fibres. Nonetheless, it is fair to say that VCS forms a ring of sphincter halves, which is in consonance with physiological results. (Fig. 1A and B). The ring extends 10–15 mm along the vestibule starting at 5–7 mm from the external orifice. Close to its cephalad and caudal limits, VCS separate ventrally then adopting a horseshoe shape (not shown). Longitudinal bundles of smooth muscle cells were localized close to deeper striated fibres (Fig. 6D).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Different vestibuli constrictor muscles?
There is little doubt that the VCS studied here increases the vestibular pressure. The timing of the twitch pressure (Pv) is close to the twitch tension wave of hind limb muscle units (Burke et al. 1971, 1973, 1974, Lewis 1972) and of the male bulbospongiosus (bulbocavernosus) muscle in vitro (Bowen et al. 1984). The tetanus/twitch tension ratio was close to that of the MG muscle (Lewis 1972).
Three different muscles had been named vestibuli constrictor, two in the cat (Crouch & Lackey 1969, Martin et al. 1974) and one in the rabbit (Martínez-Gómez et al. 1997, Cruz et al. 2002). The descriptions were based on fixed cadavers. These muscles do not resemble VCS. The vestibular constrictor described by Crouch & Lackey (1969) will be here VC1; it arises in the EAS, surrounds the vestibule up to the external orifice and inserts in the urogenital sinus. The VCS neither reaches the orifice nor does it have this insertion.
Martin et al. (1974) also stated that fascicles of EAS form a vestibuli constrictor (VC2) that is situated on the lateral wall of the vestibule but does not encircle it; the majority of VC2 fibres insert ventrally in the ischial arc. In contrast to the VC2, the VCS entirely surrounds the vestibule and the muscle fibres do not show bone insertion but end freely in the vestibule wall. Thus, neither the VC1 nor the VC2 correspond to the VCS.
The vestibuli constrictor (VC3) described in the rabbit (Martínez-Gómez et al. 1997, Cruz et al. 2002) has a bone insertion. Vaginal pressure increased by separate contraction of ICM and the bulbospongiosus muscle (BSM; Cruz et al. 2002). In the cat, the ICM does not produce Pv and the BSM was not identified in the female either by us or by Crouch & Lackey (1969) or Martin et al. (1974). Differences between species should be considered, but a sphincter similar to VCS might be present in other species. We do not believe that the cat VCS is an exception among mammals, including humans. Women's orgasm initiates with involuntary, rhythmic contractions of circumvaginal striated muscles (for review, Weston et al. 2004), and similar contractions are induced by stimulation of the clitoris (Shafik et al. 2008). We wonder whether a sphincter similar to VCS might be involved in these responses. We wonder also whether the VCS might correspond to the BSM in the male.
The magnitude of Pv or T cannot be compared with previous data from the rat (Pacheco et al. 1989) and the rabbit (Cruz et al. 2002); these authors give the lumen pressure in force units (grams), but there is no way to convert grams to cm of water.
Multiple innervation of VCS
Dogma states that a single muscle fibre receive a single motor terminal. Now, there is enough evidence to reject this dogma. (Sandmann 1885, quoted by Katz & Kuffler 1941, Jarcho et al. 1952, Rossi 1990, Zenker et al. 1990, Duxson & Sheard 1995, Happak et al. 1997, Perie et al. 1997, Lateva et al. 2002). Present results might also challenge the dogma. The sum of the unilaterally induced Pvs was larger than the Pv induced by e-MPN-LR, suggesting that some single muscle fibres might be innervated by e-MPN axons from both sides. We wonder whether this might occur in other medial muscles.
In addition, the sum of Pvs induced by maximal stimulation of e-MPN branches was larger than the Pv induced by maximal stimulation of the entire MPN. From these data, it can be inferred that some single VCS fibres might receive more than one terminal, each one inducing threshold end-plate potential (e.p.p.). In normal conditions, two or more endplates would be redundant if supplied by a single e-MPN axon that divides into daughter fibres travelling in different nerve branches. Thus, stimulating the parent e-MPN axon or each branch separately would produce muscle fibre firing (Fig. 4, diagram on the left). Alternatively, different motoneurones might innervate a single muscle fibre (Fig. 4, diagram on the right). Let us assume that a single muscle fibre receives motor terminals from both motoneurones, a and b. At a given moment, neuron a might fire but not neuron b. In another moment, neuron b might fire but neuron a does not, but at both moments the muscle fibre contracts. This would increase certainty in the motor system in spite of uncertainty in the central transmission.
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Materials and Methods |
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The iliac bone was fixed bilaterally with the cat in prone decubitus (see Cueva-Rolón et al. 1993). The trunk and branches of the pudendal was exposed in the ischial fossa on both sides of the body. In all experiments, the right pudendal nerve was sectioned close to the sciatic notch. The neighbour posterior femoral nerve (Fig. 4 in Langley & Anderson 1896), which is a cutaneous nerve, was also sectioned at the same level. The ischial fossa was covered with mineral oil that was maintained at around 37 °C by radiant heat. The left pudendal trunk was either sectioned or kept intact. The pudendal trunk divides into a sensory branch (SPN) and a motor branch (MPN). MPN divides into the internal or urethral and external branches (e-MPN), which was exposed for electrical stimulation or, in some experiments, for recording. In the present experiments, the urethral branch was sectioned distally. SPN and e-MPN were mounted on bipolar Ag electrodes for stimulation or recording.
The bulb of a number 12 Foley catheter that was tightly plugged to a pressure transducer. The bulb of the catheter was introduced as far as possible (16–21 mm) in the lumen of the vaginal vestibule (see Crouch & Lackey 1969, Watson & Glover 1993, Zambelli & Cunto 2005). The bulb (25 mm long, spindle shaped and 4 mm wide in its wider, central portion) could not be advanced further due to progressive narrowing of the vestibular lumen in the cephalad direction. The length of the vestibule is close to the length of the erect penis (Watson & Glover 1993).
Probing the vaginal vestibule
The vestibule was probed with a probe displacement of less than 1 mm (Cueva-Rolón et al. 1993). The probe was an aluminium cylinder with a probing tip 8 mm long and 6 mm in diameter covered by rubber. The entire probe was placed inside a slightly wider, hollow cylinder (the carrier) that was shorter than the probe. Thus, the probe ends emerged at both sides of the carrier, which was attached to a manipulator. A spring joining the carrier and the probe was stretched when the latter moved forward by tapping on its rear end, and recoiled after tapping. The rear end of the probe was attached to one end of the cantilever pole of a modified version of Statham strain gauge (Delgado-Lezama et al. 1997). A mirror attached perpendicularly to the mobile cantilever pole reflects the light supplied by an optocoupler (OC) fixed to the strain gauge and connected to a constant current amplifier. The OC sends a light beam to the mirror, and converts linearly the reflected light to output voltage; reflected light depends linearly on the distance between the mirror and the OC beam. The OC output was connected to the recording instrument. The probe tip was introduced in the vestibule using the manipulator holding (the carrier). A 4 mm wide pole of a plastic material was attached to the movable centre of a modified load speaker (electromagnetic coil; EMC), which was held to a manipulator and fed with a conveniently amplified square pulse. The free end of the EMC pole was concave, and it was positioned in close contact with the complementary convexity of the transducer free end pole, which moves the strain guage contilever. Therefore, any movement of the EMC pole was transmitted to both the transducer pole and the vaginal probe and vice versa. The probe movement was close to a half-sinusoidal wave. Thus, the probing device consists of the in-series arrangement of probe, the modified strain gauge and the EMC. The strain gauge output was used to monitor the probe movement. Additional details are given in the results.
At the end of the experiments, the cats were killed by an overdose of pentobarbitone.
Statistical analysis
Automatic average (N=30–100) of the pressure wave (Pv) induced by single electric shocks was used to estimate the peak pressure, the time to peak, decay time to half the peak pressure an area under the pressure wave using a software program (Origin 5.0). The S.E.M. of the average was used to estimate the variability of these parameters; the S.D. of these parameters in the samples of each average was less than 5%. The t-test was used to find statistical significance between paired averages.
VCS structure
Just after killing the cat, the vestibule with part of the distal vagina was excised and fixed either with 1.25% glutaraldehyde in 0.1 M cacodylate solution for electron microscopy (EM) or light microscopy, or with buffered 4% paraformaldehyde for light microscopy. The fixed vestibule was transversally divided with scalpel into 5 mm thick blocks that were photographed. Transverse slices 5 µm thick embedded in paraffin were stained with haematoxylin–eosin for light microscopy. Specimens of about 1 mm3 post-fixed with osmium were processed for epoxy embedding. Transversal or longitudinal semi-thin sections were used for light microscopy staining with toluidine blue or with uranyl acetate and lead citrate for EM. Additional details are given in the section of results.
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Declaration of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Funding |
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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Received August 4, 2008
First decision September 18, 2008
Revised manuscript received October 29, 2008
Accepted November 21, 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestAcknowledgementsReferences |
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