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Motor pudendal nerve characterization in the female rat
Article first published online: 5 DEC 2001
Copyright © 2002 Wiley-Liss, Inc.
The Anatomical Record
Volume 266, Issue 1, pages 21–29, 1 January 2002
How to Cite
Kane, D. D., Shott, S., Hughes, W. F. and Kerns, J. M. (2002), Motor pudendal nerve characterization in the female rat. Anat. Rec., 266: 21–29. doi: 10.1002/ar.10029
- Issue published online: 7 DEC 2001
- Article first published online: 5 DEC 2001
- Manuscript Accepted: 3 OCT 2001
- Manuscript Received: 25 APR 2001
- external anal sphincter;
- external urethral sphincter;
- Onuf's nucleus;
- fluorescent tracers;
- axon branching
The aim of our study was to provide quantitative data on pudendal motor neuron cell bodies and axons in the female rat. To confirm earlier studies, fluorescent retrograde tracers were used to label the motor neurons for correlation with myelinated axon counts along the length of the motor pudendal nerve. The external urethral sphincter of female rats was injected with diamidino yellow and the external anal sphincter with fast blue. The L6 spinal cord revealed labeled motor neurons. Those in the dorsolateral column (60.8 ± 10.6) had nuclei labeled yellow from the external urethral sphincter and those in the dorsomedial column (31.7 ± 8.5) had cytoplasm labeled blue from the external anal sphincter. Double labeling was not present, suggesting that pudendal motor neurons in each column innervate separate sphincters. The motor pudendal nerve in the ischiorectal fossa was also characterized by light microscopy. The mean myelinated axon count (151.4 ± 17.0) was highly correlated (r = 0.995) in the proximal fascicles and the sum of distal fascicles. This indicated that myelinated axons do not branch at the point where the main motor pudendal nerve branches into separate fascicles. Axon counts between sides were not as well correlated (r = 0.883). The ratio of motor neurons to myelinated axons is 56%, suggesting that some myelinated axons either innervate other muscles or are sensory. This reproducible characterization of the normal pudendal nerve anatomy provides an excellent basis for experimental studies associated with pudendal nerve denervation as a model for neurogenic incontinence. Anat Rec 266:21–29, 2002. © 2002 Wiley-Liss, Inc.
Damage to the pudendal nerve in the clinical setting can lead to urinary and/or fecal incontinence (Allen et al., 1990; Meyer et al., 1998; Ryhammer et al., 1995; Snooks et al., 1984, 1985). From the clinical perspective, the high incidence of incontinence in postpartum and postmenopausal women might be due in part to non-neural factors affecting muscle and connective tissue (Delancy, 1996; Smith, 1994) or vaginal distention (Lin et al., 1998). The need, however, to assess the role of nerve related damage or aging should not be underestimated. A complete characterization of the pudendal nerve is needed in an animal model to better understand the neural changes associated with incontinence (Heidkamp et al., 1998; Kerns et al., 2000; Lin et al., 1998; Sakamoto et al., 2000). A high degree of pattern uniformity would further strengthen the utility of the model.
Pudendal motor neurons in the human are located in a single column in the sacral cord called Onuf's nucleus (Onufrowicz, 1901; Pullen et al., 1997; Schrøder, 1981). The equivalent pudendal motor neurons in the rat are in two separate columns, which are sexually dimorphic and located in the L6 spinal cord (Breedlove and Arnold, 1980; Goldstein and Sengelaub, 1992; McKenna and Nadelhaft, 1986). The external urethral sphincter (EUS) neurons are located in the dorsolateral (DL) column, whereas the external anal sphincter (EAS) is innervated by others in the dorsomedial (DM) column (Katagiri et al., 1986; McKenna and Nadelhaft, 1986; Schrøder, 1980; Ueyama et al., 1987). There are additional motor neurons in both columns for sexual reflexes. Distinct features of the cells in comparison to other motor neurons include prominent dendritic bundles (Ding et al., 1995; Kerns and Peters, 1974; McKenna and Nadelhaft, 1986) and adult expression of p75NGFR and GAP-43 (Koliatsos et al., 1994; Nacimiento et al., 1993).
Other investigators have shown that the rat pudendal nerve has separate motor and sensory branches (McKenna and Nadelhaft, 1986; Pacheco et al., 1989; Ueyama et al., 1987). As the motor branch of the pudendal nerve (PN) courses through the ischiorectal fossa (IRF), it bifurcates into a dorsal EAS fascicle and a ventral EUS fascicle. These fascicles continue to subdivide as they approach their respective targets. It is not known whether individual motor neurons send branches to both sphincter targets. Double labeling methods have shown such branching of single axons in other systems (Cheng and Powley, 2000; Hisa et al., 1984; Russo and Conte, 1996).
Some investigators have shown axon branching occurring within the peripheral nerves at a location just proximal to the muscle target, but also along the midpoint in the case of the cranial nerves to the extraocular muscles (Sunderland and Lavarack, 1953). This has been suggested by studies where one ulnar motor neuron sends one axon that branches to innervate two separate muscles (Sunderland, 1968). Sensory axons in the cutaneous nerves must also branch along the distal course because their fiber numbers increase (Lavarack et al., 1951).
It is also well known that the intraneural fascicular topography is complex, with extensive bundle rearrangements (Sunderland, 1968). This concept has been challenged by Jabaley et al. (1980), but more recent evidence with retrograde tracers indicates that functional units remain in individual sectors of the nerve over long distances (Brushart, 1991). It is still not clear, however, whether myelinated axons in the extramuscular nerve also branch when the fascicle branches. This important question is addressed in the present study because absolute fiber counts at several levels can be easily and accurately determined.
In the present study, we confirmed the description of McKenna and Nadelhaft (1986) that pudendal motor neurons have distinct origins in the ventral spinal cord and determined that their myelinated axons traverse the peripheral pathway to their separate sphincter targets without branching. We used retrograde tracers to label motor neurons and counted the myelinated axons at three locations in the IRF, respectively. Our aim was to study the PN origins, peripheral pathways, and striated sphincter innervation in the female rat to establish the neural components for use in an experimental model of stress urinary incontinence. This study has been presented earlier in abstract form (Kane et al., 2000).
MATERIALS AND METHODS
Twenty-one virgin female Sprague–Dawley rats (200–250 g) from Harlan Industries (Indianapolis, IN) were maintained with a 12 hr light/dark cycle. Animal care was in accordance with institutional and federal guidelines.
Fluorescent Tracer Study
Animal surgery and injection.
Rats were anesthetized with an intraperitoneal injection of Ketamine (90 mg/kg body weight) and Xylazine (5 mg/kg body weight). A preliminary study was done on three animals using horseradish peroxidase (HRP, 30% aqueous, Sigma VI, Sigma Chemical Co., St. Louis, MO) to determine the optimal injection sites for each sphincter. The post-injection survival time was 3 days. Seven rats had the two fluorescent tracers injected into separate sphincters. The first injection, 18–21 μl of Diamidino Yellow ([DY, Sigma Chemical Co.], 2% aqueous in 4% DMSO), was injected into the surgically exposed EUS on both lateral sides and ventromedially. Hemostasis was obtained and the skin incision closed with sutures. This was followed by percutaneous injections totaling 18–22 μl Fast Blue ([FB, Sigma Chemical Co.] 2% aqueous) in the dorsomedial, ventromedial, and both lateral sides of the EAS. Two additional rats had the tracer injections reversed, to compare the labeling characteristics of the two tracers. Using the same procedure and tracer quantities, DY was injected in the EAS and FB into the EAS. To confirm double labeling, one animal had only the EAS injected with both FB and DY via separate syringes in concentrations above. After recovery from anesthesia, the animals were given buprenorphine (0.05 ml) for pain relief.
Spinal cord removal and sectioning.
At 6 days after injection, the animals were anesthetized and fixed by cardiac perfusion. After a heparin (0.1 ml) injection into the left ventricle, a saline washout was followed with 350 ml of fixative (4% paraformaldehyde in 0.1 M phosphate buffer). The spinal cords were removed and trimmed to include the rostral L4 and caudal S2 regions. The ventral surface of the spinal cord was sectioned horizontally on a vibratome (Lancer series 1000) at either 50 or 100 μm. Pudendal motor neurons were identified in several sections under a dissecting microscope using transillumination and the sections were serially mounted on slides.
Motor neuron analysis using fluorescent microscopy.
Using a fluorescent microscope (Leitz Orthoplan, 360-barrier filter, 40×), the labeled motor neurons were directly counted three times to ensure precision. Motor neurons were categorized as DL, DM, or “other.” The FB labeled cell body was not distinct, so HRP labeled cell bodies of both the EAS and EUS motor neurons were measured from camera lucida tracings. The DY labeled nucleus of the EAS and EUS motor neurons was measured from photomicrographs. The major and minor axis measurements were averaged in obtaining either the cell body or nuclear diameter. The sizes were used in the Abercrombie correction factor (Abercrombie, 1946) to prevent double counting.
Anatomy of the Motor Pudendal Nerve
Nerve dissection and low power characterization.
A total of 24 nerves from 14 rats were examined, including six from the fluorescent tracer experiment. In 10 animals the nerves were compared on both sides. As stated above, the total number of animals in the entire study was 21. Fixation was by cardiac perfusion with a saline wash, 150 ml of dilute (1.25% glutaraldehyde/1% paraformaldehyde) fixative, and 300 ml of concentrated (5% glutaraldehyde/4% paraformaldehyde) fixative. The IRF was dissected to obtain the neurovascular sheet, which was stained with aqueous 2% osmium tetroxide for 45–60 min. The motor pudendal nerve branching patterns were characterized from the osmicated whole mount specimens. Branching fascicles were counted from both the EAS and EUS branches. The distance from the EAS/EUS branch point to the first fascicle branch point of both EAS and EUS branches was measured and photographed.
Nerve sectioning and staining.
The pudendal nerve sheet was cut into three pieces, using the EAS/EUS branch point as a reference (Fig. 1). The proximal one segment (P1) was cut 6–8 mm proximal to the EAS/EUS branch point, whereas the proximal two segment (P2) was 1.5–2 mm proximal to the EAS/EUS branch point. The distal segment (D) was just distal to the EAS/EUS branch point and it contained both the EUS and EAS branches. The three individual segments were further stained en bloc with 1% uranyl acetate, dehydrated in alcohol rinses, and embedded in Epon-Araldite. The nerve segments were transversely sectioned at 1 μm, using a LKB Ultratome V and the sections were stained with methylene blue-azure II.
Myelinated axon counts.
Using an ocular grid, all visible myelinated axons were counted by the same investigator three times to determine a mean. Small fascicles were usually present in the distal segment and were included as part of either the EAS or EUS, depending on their proximity to either branch.
The number of labeled neurons in the first experiment, as well as the number of EAS/EUS fascicles, the fascicle branching distance, and motor pudendal nerve myelinated axon counts from the second experiment were obtained and expressed as the mean ± SD. A paired t-test (SPSS 10 for Windows 98) was done to compare the motor neuron counts from the right and left side. Repeated measures ANOVA and Pearson correlation coefficients (SPSS 10 for Windows 98) were used to compare myelinated axon counts between segments and sides. In both cases, P < 0.05 was used to indicate a significant difference. Original slides for montages were scanned and the images were processed in Adobe Photoshop (v. 5.0) to enhance contrast for publication.
Fluorescent Tracer Study
Labeling of motor neurons.
The EAS injection resulted in motor neurons labeled in the DM column and the EUS injection in motor neurons in the DL column of the L6 spinal cord segment. A few DM or DL motor neurons were labeled in the caudal portion of L5 and the rostral portion of S1. The distance between the DL and DM columns was approximately 550 μm, which was similar to other studies (McKenna and Nadelhaft, 1986; Ueyama et al., 1987). The “other” cells were those labeled outside the DL and DM motor neuron columns, indicating that other muscles incorporated the tracer. Eight of nine animals had very few cells (<8) labeled outside the DL or DM columns. Due to their location and relatively small numbers, these cells did not impact the pudendal motor neuron counts.
Double labeling was not present in any of the cell groups. FB labeled the cytoplasm and dendrites blue (Fig. 2a). There was no prominent FB nuclear labeling observed. DY labeled the nucleus a fluorescent yellow color (Fig. 2b). There was cytoplasmic labeling in some motor neurons labeled with DY, but the cytoplasm was a dull yellow rather than royal blue as seen in cells labeled with FB. Glial cell nuclei in a perineuronal area were abundantly labeled with the DY tracer (Fig. 2b), but this was not observed when motor neurons were labeled with FB.
There was double labeling present in the animal that had both tracers injected into the EAS (Fig. 2c). DY was present in the nucleus and nucleolus, whereas FB was present in the cytoplasm and dendrites. Glial nuclei were present and labeled with DY only.
Fluorescent labeled motor neuron counts.
The mean number of motor neurons per side was 60.8 ± 10.6 in the DL column, 31.7 ± 8.5 in the DM column, and 5.8 ± 12.8 for “other” cells (Table 1). There were almost twice as many motor neurons in the DL column compared to the DM column. Using a paired t-test, there were no significant differences in counts from the right or left sides.
|Other left||1.8||2.9||0.0– 8.7||9|
|Breedlove and Arnold, 1980*|
|McKenna and Nadelhaft, 1986*|
|Ueyama et al., 1987*|
|Kerns et al., 2000|
One animal had the EAS injected with both FB and DY as a control. The mean number of DM motor neurons labeled was 31.2, but of that number 22.5 were double labeled, 6.4 labeled with FB only, and 2.3 with DY only. Labeling was observed only in the L6 spinal cord segment in this control animal.
Motor neuron and nuclear diameters.
Using HRP labeled cells, the cell body size of DL and DM motor neurons was obtained. The DL (EUS) mean diameter was 24.4 ± 3.0 μm (25 neurons, 1 animal) and the DM (EAS) was 25.0 ± 3.7 μm (26 neurons, 2 animals). Using diamidino yellow, the nuclear measurement from the DL column was 16.2 ± 1.2 μm (20 neurons, 4 animals) and the DM column 19.5 ± 1.9 μm, (20 neurons, 2 animals). The size uniformity corresponded with reported values of pudendal motor neurons (McKenna and Nadelhaft, 1986; Ueyama et al., 1987) and our values were used in the Abercrombie correction factor to prevent double counts.
Anatomy of the Motor Pudendal Nerve
Motor pudendal fascicle branching.
The motor pudendal nerve was a single fascicle as it traveled from the lumbosacral plexus into the ischiorectal fossa. It then bifurcated into a superficial and a deep fascicle (Fig. 1), which we understood to be branches to the EAS and EUS, respectively (McKenna and Nadelhaft, 1986). In a few cases, there was minor branching associated with the P1 area. These small fascicles, however, would rejoin either the P2 segment or the D segment before the EAS/EUS fascicle bifurcation.
The EAS fascicle (N = 22) either branched into two (95%) or three (5%) smaller fascicles at a mean distance of 3.8 ± 0.8 mm (range 3.0–5.0 mm) from the EAS/EUS branch point. The EUS fascicle (N = 27) branched into two (26%), three (67%) or four (7%) fascicles with a mean branching distance of 1.0 ± 0.4 mm (range 0.3–1.5 mm) from the EAS/EUS branch point.
Cross sectional anatomy of the P1, P2, and D segments.
The typical light microscopic appearance of the three segments in transverse view is shown in Figure 3. The myelinated axons from the left side were counted three times and averaged (Table 2). The P1 segment contained 150.9 ± 18.3, P2 segment 150.9 ± 18.6, and D segment 150.8 ± 17.1 myelinated axons. The D segment mean myelinated axon counts were divided into an EUS fascicle 89.3 ± 12.8 and an EAS fascicle 61.4 ± 7.5.
The myelinated axons from the right side in the P1 segment contained 152.2 ± 17.3, P2 segment 151.7 ± 16.7, and D segment 152.1 ± 17.4 myelinated axons. The D segment mean myelinated axon counts were divided into an EUS fascicle 92.3 ± 12.1 and an EAS fascicle 59.8 ± 13.5. The overall mean number of myelinated axons from all three segments on both sides was calculated as 151.4 ± 17.0 (Table 2).
Same-side and opposite-side comparisons.
Using repeated measures ANOVA, there were no significant differences in the myelinated axon counts between either location (P1, P2, D) or side (R, L). Using the data from the myelinated axon counts, comparisons were made between locations (P1, P2, D) from the same and opposite sides (Fig. 4) of the pudendal nerve. On the left side, the Pearson correlation coefficient range for comparing location was between r = 0.995–0.996 (P < 0.0005) (Fig. 4a) and on the right r = 0.996 (P < 0.0005). When comparing left vs. right side locations, however, the Pearson correlation range was between r = 0.853–0.883 (P ≤ 0.02) (Fig. 4b). The correlation coefficients for all combinations are given in Figure 4c.
Comparison of Results
In Table 3 we show data from four animals that overlapped both experiments. A ratio of the pudendal motor neurons/myelinated axon numbers shows more individual variation when examining the separate sides (range = 42.3–67.6%) compared to the average of the two sides (range = 54.7–58.6%). Overall, there are roughly half as many pudendal motor neurons as myelinated axons.
|%*||42.3||57.1||48.8||56.2||51.0 ± 7.0|
|%*||67.6||56.1||61.5||61.0||61.4 ± 4.7|
|%*||54.7||56.6||54.9||58.6||56.2 ± 1.8|
The present study confirms that the pudendal motor neurons in the L6 spinal cord of the female rat have distinct origins in the dorsolateral and dorsomedial columns, which supply the external urethral and external anal sphincters, respectively. This conclusion is based on the segregation of label to each column (DL and DM) after injection of the two target muscles (EUS and EAS) with a different retrograde tracer (DY and FB). Furthermore, precise myelinated axon counts at three locations along the motor pudendal nerve have been taken and indicate the axons do not branch in their peripheral pathway through the ischiorectal fossa, even though the nerve fascicles subdivide. These two parameters may be useful in evaluating regeneration/degeneration changes that occur in the pudendal nerve after injury.
Fluorescent Tracer Study
Although previous studies have used HRP (Gerrits et al., 1997; McKenna and Nadelhaft, 1986; Ueyama et al., 1987), fluorescent double labeling has been used in other systems (Bentivoglio et al., 1980; Keizer et al., 1983; Kuypers et al., 1980; Kuzuhara et al., 1980). FB and DY have been used to study the pudendal system in the rat (Russo and Conte, 1996; Kerns et al., 2000). Counts and location of FB/DY labeled pudendal motor neurons in our experiment generally agree with those from other studies (Breedlove and Arnold, 1980; Kerns et al., 2000; Marson, 1997; McKenna and Nadelhaft, 1986; Schrøder, 1980; Ueyama et al., 1987). The number of labeled motor neurons in the DL column was almost twice that found in the DM column. To fully explain the difference in DL/DM motor neuron number, the number of motor endplates, muscle fibers, and motor unit size is needed.
Technical factors might influence the results of cell counting (Collins et al., 1991; Cheng and Powley, 2000). In the present study, FB was localized in the cytoplasm whereas DY localized in the nuclei, as shown in Figure 2c. This feature would have permitted the clear detection of double-labeled cells, had they been present. Because FB generally labels 25% fewer cells after a 4-day survival as compared to HRP (Aschoff and Hollander, 1982; Illert et al., 1982), our FB counts may be an underestimate of the motor neuron numbers.
Anatomy of the Motor Pudendal Nerve
Our study confirms early reports that the motor pudendal nerve bifurcates into separate fascicles that innervate the EAS and EUS (McKenna and Nadelhaft, 1986; Pacheco et al., 1989, 1997; Ueyama et al., 1987). Within the proximal ischiorectal fossa, the internal obturator nerve separates the sensory and motor pudendal nerves. A profuse fascicular branching pattern occurs more distally, which has not been previously reported. More studies, however, are required to determine the precise terminal branching patterns in the targets.
The significance of myelinated axon counts in the pudendal nerve may relate to considerations of gender (Moore and White, 1996), nerve type (Hulsebosch and Coggeshall, 1982), and development (Hanzlikova and Gutmann, 1972). We observed differences in myelinated axon counts between sides and between animals. A low myelinated axon number on one side did not, however, appear to be compensated by a higher number on the contralateral side. Terminal field branching could compensate for the observed variation in myelinated axon counts to ensure that the sphincters are appropriately and sufficiently innervated for normal function.
Axonal branching can theoretically occur at any point along the nerve pathway, e.g., near the spinal cord (Fraher and O'Sullivan, 1989; Langford and Coggeshall, 1981; Pierau et al., 1982; Taylor and Pierau, 1982), or in terminal fields (Kerns, 1980; Pfeiffer and Friede, 1985). The middle region, however, has not been adequately studied to provide a basis for evaluating pudendal nerve lesions. Our myelinated axon counts in an 8 mm motor pudendal nerve segment within the ischiorectal fossa indicate that branching did not occur, despite the branching of the nerve fascicle (Fig. 2). Although most motor neurons supply a single muscle, it is known that a motor neuron can either supply two separate muscles through a single nerve (Hisa et al., 1984) or can send myelinated axon branches through two separate nerves (Yajima and Hayashi, 1989). In accordance with the more common arrangement, the absence of double-labeled pudendal motor neurons in our study confirms that these axons do go to one or the other, but not both sphincters. This pathway is accompanied by fascicular re-arrangements and branching in the distal EAS and EUS segments. A recent review addresses the several mechanisms that might control axonal branching (Acebes and Furrús, 2000), but whether the same controls apply to fascicular branching has not yet been determined.
Based upon the quantitative data from both parts of our study (Table 3), it is apparent that the number of myelinated axons exceeds that of motor neurons. One might expect a 1:1 ratio unless either there was branching at a more proximal location or motor neurons were unlabeled. We believe, however, that these “extra” axons are either motor axons to muscles other than the sphincters or are sensory axons. Some small myelinated preganglionic autonomic fibers possibly travel within the pudendal nerve, although most of these are believed to travel within the pelvic nerve.
On the other hand, several studies have reported that extra-pudendal fibers may contribute to innervation of the sphincters or pelvic floor musculature in the human (Borirakchanyavat et al., 1997; Juenemann et al., 1988; Zvara et al., 1994) and the dog (Tanagho et al., 1982). Our previous results do not exclude this possibility in the rat (Kerns et al., 2000), where a small number of motor neurons (14.5/78.2 = 18.5%) to the EUS could be extra-pudendal. Future studies incorporating anatomical, molecular and electrophysiological methods (Kerns et al., 2000; Lin et al., 1998; Sakamoto et al., 2000) may help resolve the importance of extra-pudendal innervation.
In summary, our study clearly defines consistent anatomical landmarks that insure accurate motor pudendal axon counts. We also suggest that a segment proximal to a lesion of the pudendal nerve would provide a better control reference than the contralateral nerve. Our work confirms earlier tracer study results on the number of pudendal motor neurons. The direct counts of motor neurons and axons provide manageable numbers, which allow better interpretation of treatment outcomes. The principal findings of motor neuron numbers in relation to axon counts at the sites designated provide an excellent reference for looking at fiber regeneration or cell loss after pudendal nerve lesions. The normal ratio of 56% for motor neurons/myelinated axons could be altered in several ways at a site distal to the injury: 1) the cell numbers could decrease as a result of cell loss or remain the same; 2) axon numbers could decrease due to a failure of regeneration; or 3) axon numbers may increase as a result of sprouting at the lesion site. Each of these changes could contribute to deviations from the normal ratio determined in the present study. The quantitative measures that best relate to a threshold for continence is presently unknown.
The descriptive and quantitative information from our study is required in regeneration studies involving pudendal nerve lesions as a model for neurogenic incontinence. A more complete understanding of axon branching and motor unit size in the urethra will also provide a basis for understanding neural reorganization after injury in this model. These insights may apply to neurogenic components of incontinence in the clinical setting after pudendal nerve injury in the human.
We thank Drs. M.S. Damaser and L. Brubaker for their input and encouragement during the course of this study, which was in partial fulfillment for the Master's degree (D.D.K.).
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