Functional and neuroanatomical effects of vaginal distention and pudendal nerve crush in the female rat

PURPOSE: We tested the hypothesis that neuroanatomical degeneration near the external urethral sphincter (EUS) would parallel urinary dysfunction after vaginal distention or bilateral pudendal nerve crush in female rats. MATERIALS AND METHODS: A total of 28 female rats underwent bilateral pudendal nerve crush or vaginal distention, or were unoperated controls. Two days later a catheter was implanted into the bladder dome and 2 days after that (4 days after injury) urethral leak point pressure testing was performed with the rat under urethane anesthesia. The pudendal nerve and urethra were then dissected and prepared for light and electron microscopy. RESULTS: Leak point pressure was significantly decreased 4 days after pudendal nerve crush and vaginal distention (29.3 +/- 3.4 and 31.0 +/- 2.5 cm H(2)O, respectively) compared with controls (44.3 +/- 3.4 cm H(2)O). The percentage of nerve fascicles with degeneration near the EUS was significantly greater in the nerve crush (13.1% +/- 1.7%) and vaginal distention (7.2% +/- 2.2%) groups than in the control group (0% +/- 0%). There were fewer nerve fascicles near the EUS in the ventral half of the urethral cross section than in the dorsal half in all 3 groups and the percent of fascicles with degeneration was greater in the ventral half than in the dorsal half in the 2 injury groups. CONCLUSIONS: These results suggest that the pudendal nerve is particularly vulnerable to injury during vaginal distention in this animal model. The 2 injury models may be useful for investigating the pathophysiology of stress urinary incontinence.     

The histochemical study of rat sciatic nerve cholinesterase in degeneration and regeneration

With a modified, less time-consuming, Karnovsky-Roots method, this study evaluated the reduction and recovery of rat sciatic nerve cholinesterase (ChE) in degeneration and regeneration. Characteristics of motor fascicles included not only partial staining of acetylcholinesterase (AchE) in the axon, but also a small amount of more intense staining of butyrylcholinesterase (BuchE) in the unmyelinated fibers, scattered between the myelinated fibers in a spot or fleck form. Characteristics of sensory fascicles included more intense staining of BuchE in the unmyelinated fibers, (appearing as irregular balls or pieces), and a scattering between unstained myelinated fibers. Mixed fascicles had characteristics of both motor and sensory fascicles. Unmyelinated fibers exhibited BuchE activity, but myelinated fibers exhibited AchE activity. Preparation time for a specimen to react to ChE-positive staining was about 1 to 2 hr; the method is very suitable for diagnosis during surgery. After nerve transection, AchE activity at the distal nerve end began to reduce gradually; no AchE could be tested after two weeks. But BuchE activity in unmyelinated fibers could be found until 30 days after transection. After epineurial suturing of the peripheral nerve transection, new AchE activity could be found at the anastomosis site until about two weeks and at 1 cm distal to the anastomosis site until about three weeks. It became more intense with the passage of time and, at about six weeks, regenerated AchE-positive myelinated fibers could be seen at the distal end of the nerve.     

Correlation between target reinnervation and distribution of motor axons in the injured rat sciatic nerve

Peripheral nerve injuries are rarely followed by complete return of function. Deficits are particularly important for motor function, resulting in paralysis and muscle atrophy. In different groups, the sciatic nerve was either crushed or transected and repaired by direct suture or by tube repair using silicone or collagen tubes. After 60 days, nerve regeneration was assessed by electrophysiological and functional tests, nerve morphology and immunohistochemistry against choline acetyltransferase (ChAT) for labeling motor axons. Suture and tube repair resulted in similar levels of muscle reinnervation, but significantly lower than after nerve crush. Recovery of walking track pattern was poor in all groups after nerve section. The numbers of regenerated myelinated fibers and of ChAT+ fibers were similar to control values after nerve crush, but increased after section and repair. The normal fascicular architecture and grouping of ChAT+ fibers were maintained after nerve crush, but lost after section and repair, where motor fibers were scattered within small regenerated fascicles throughout the nerve. The loss of fascicular organization was related to the deficient recovery of locomotor function. Thus, labeling of motor axons by ChAT immunohistochemistry provides useful information for the study of the degree and specificity of nerve regeneration.     

Functional evaluation of regenerated and misrouted low threshold mechanoreceptors and polymodal nociceptors in the skin of rat hindfeet after crush lesions to the sciatic nerve.

Tracer studies on motor axons after nerve crush injuries have indicated that misrouting may occur even when the endoneurium is intact. Misrouting of regenerated polymodal nociceptive C-fibres and low threshold mechanoreceptive axons have been studied functionally in 50 rats three months after unilateral crush lesions to the sciatic nerve. Two weeks before evaluation the tibial fascicle (or the peroneal fascicle) above the lesion was cut and tied off. In this way only functional regeneration of misrouted axons was tested. Misrouted low threshold mechanoreceptive axons and polymodal nociceptor C-fibres were found after regeneration in both glabrous and hairy skin. We conclude that functional misdirection of both myelinated and unmyelinated sensory axons innervating either glabrous or hairy skin can occur after a crush lesion to a peripheral nerve in rats.     

Carbonic anhydrase histochemistry. A potential diagnostic method for peripheral nerve repair

Many large-diameter myelinated axons in spinal dorsal roots contain carbonic anhydrase activity, whereas few small-diameter ventral root axons stain for this enzyme. This differential localization of carbonic anhydrase in sensory and motor nerve fibers is indicative of the potential of carbonic anhydrase histochemistry to provide a convenient method for identifying predominantly motor or sensory fascicles in cut ends of peripheral nerves, thereby facilitating coaptation of fascicles in peripheral nerve repair.     

内脏神经-体神经端侧吻合后神经纤维再生研究

目的 观察大鼠内脏神经-体神经端侧吻合后神经纤维的再生.方法 24只成年SD大鼠随机分为实验组(n=12)和正常对照组(n=12),实验组大鼠通过内脏神经-体神经端侧吻合建立人工体神经-内脏神经反射弧6个月后,在吻合口近端和远端分别截取10 mm的供体神经(L4VR)和受体神经(L6VR),在L6VR延续的盆副交感神经(PPN)和阴部神经(PN)分别截取10 mm的神经.正常对照组大鼠分别取相应节段的L4VR、L6VR、PPN和PN神经.标本经石蜡包埋切片并行甲苯胺蓝染色,比较实验组和对照组大鼠L6VR、PPN、PN神经纤维数量.结果 实验组大鼠横断面可见新生的有髓神经纤维,L4VR、L6VR、PPN和PN的神经纤维数量分别为1602.2±75.7、1037.9±123.6、817.0 ±52.2、510.4±29.1,吻合口远近端神经纤维通过率为64.8%,实验组和对照组大鼠相应的L6VR、PPN、PN神经纤维数目比率分别为70.2%、68.9%和62.2%.结论 大鼠内脏神经-体神经端侧吻合后体神经能够长入并替代内脏神经.     

Nerve fibre composition of the palmar cutaneous branch of the median nerve and clinical implications.

Fifteen fresh human cadaver hands were dissected, using x2.8 loupe magnification, to study the subcutaneous innervation at the site of the incision (in the line with the radial border of the ring finger) for standard open carpal tunnel decompression. Subcutaneous nerve branches were detected and traced proximally to determine their origin. Morphometric analysis of nerve cross sections from the site of the incision and from the main nerve trunk proximal to cutaneous arborisation was performed using light and transmission electron microscopy and a computer-based image analysis system. At the site of the incision, the ulnar sub-branch (US) of the palmar cutaneous branch of the median nerve (PCBMN), which innervates the skin over the hypothenar eminence, was found in 10 of 15 cases. Branches from the ulnar side were not detected. The main trunk of PCBMN consisted on average of 1000 (SD 229) myelinated axons arranged in 1-4 fascicles. In the US of the PCBMN there were on average 620 (SD 220) myelinated axons, 80% of them smaller than 40 microm(2) i.e. thin myelinated axons, and on average 2037 (SD 1106) unmyelinated axons, arranged in 1-3 fascicles. The ratio of the number of myelinated axons in the US and the main trunk of the PCBMN was on average 63% (SD 19%). Frequency distribution of cross-sectional areas of myelinated axons shows no significant difference between the US and the main nerve trunk of the PCBMN. The importance of incision trauma to subcutaneous innervation of palmar triangle is emphasised and possible mechanisms of scar discomfort are discussed.     

人体膈神经与副膈神经的组织化学研究

研究人体膈神经与副膈神经的运动纤维数量及有髓纤维截面积。方法：运用Ｋａｍｏｖｓｋｙ－Ｒｏｏｔｓ［１］乙酰胆碱酯酶组织化学方法，研究１２例成人膈神经及并存的５例副膈神经。对轴索内酶活性反应呈阳性的纤维，进行计数及纤维截面积图象分析。结果：膈神经与副膈神经绝大部分有髓纤维酶反应呈阳性，所含阳性有膈纤维分别为２６８６根、６３４根。膈神经与副膈神经并存时有髓纤维总数达３２１８根。有髓纤维截面积分别为１０３．８μｍ２、９４．７μｍ２。结论：膈神经含２６８６根运动纤维，有髓纤维截面积为１０３．８μｍ２，是良好的移位动力神经；副膈神经与其并存，有重要的临床意义。     

神经移植段对神经再生影响的实验研究

为了探讨神经移植段的方向对神经再生的影响，采用硅胶管桥接Ｗｉｓｔａｒ大鼠双侧坐骨神经，一侧硅胶管远端套接顺行神经移植段，另一侧套接逆行神经移植段，术后２，４及６周取材。     

Structural and functional investigations of the murine cavernosal nerve: a model system for serial spatio-temporal study of autonomic neuropathy

OBJECTIVE: To illustrate the ultrastructural fibre composition of the rat cavernosal nerve at serial levels, from its origin in the main pelvic ganglion to its termination in the corpus cavernosum of the distal penile shaft, and to develop a technique that permits repeated electrophysiological recording from the fibres that form the cavernosal nerve distinct from the axons of the dorsal nerve of the penis (DNP). MATERIALS AND METHODS: For the light microscope and ultrastructural studies, Sprague-Dawley rats were anaesthetized and the pelvic organs and lower limbs were perfused with glutaraldehyde through the distal aorta. Tissue samples were embedded in epoxy resin and prepared for light and electron microscopy. Frozen tissue was used for the immunohistochemical studies and sections were stained with rabbit anti-nitric oxide synthetase 1 (NOS1). For the electrophysiology, anaesthetized rats were used in sterile conditions. Nerve conduction velocity for the cavernosal nerve was assessed from a point 2 mm below the main (major) pelvic ganglion after stimulating the nerve at the crus penis; multi-unit averaging techniques were used to enhance the recording of slow-conduction activity. Recordings from the DNP were obtained over the proximal shaft after stimulation at the base of the penis. RESULTS: Step-serial sections of the cavernosal nerve revealed numerous ganglion cells in the initial segments and gradually fewer myelinated fibres at distal levels. At the point of crural entry, the nerve contained almost exclusively unmyelinated axons. As it descended the penile shaft, the nerve separated into small fascicles containing only one to four axons at the level of the distal shaft. In the corpus cavernosum, vesicle-filled presynaptic axon preterminals were close to smooth muscle fibres, but did not seem to be in direct contact. Immunohistochemical evaluation of NOS1 activity showed intense staining of the fibres of the DNP and most of the neurones in the main pelvic ganglion. There was also scattered NOS1 activity in the nerve bundles of the corpus cavernosum. Electrophysiology identified activity in C fibres on the cavernosal nerve and in Aalpha-Adelta fibres in the DNP. CONCLUSION: These results show that it is possible to perform integrated cavernosal pressure monitoring and ultrastructural and electrophysiological studies in this model. These yielded accurate data about the erectile status of the penis, and the state of unmyelinated and myelinated fibres in the DNP and cavernosal nerves of the same animal. This study provides a useful template for future studies of experimental diabetic autonomic neuropathy.     

神经再生室中神经再生过程和微环境的研究

目的 探讨再生室中神经再生过程、微环境成份及作用。方法 取SD大鼠70只,坐骨神经于股中部切断,用硅胶管套接,神经断端间间距10mm。于术后3d,1、2、3、4、5、6周取材,将10mm再生段由近向远分为S1,S2,S3,S4,S5,共5段,每段2mm,行组织学和电镜观察。结果 术后3d,再生充满棕黄色浑浊液体。术后1周,再生室中央形成条状连接,巨嗜细胞迁移至S3会合。术后2周成纤维细胞、雪旺细胞、内皮细胞迁移到S3会合。有髓神经纤维长入S1,无髓神经纤维长入S3。术后3周无髓纤维通过再生室,有髓纤维长入S3。术后4周有髓纤维通过再生聚集成束。术后5、6周神经纤维数量增多,髓鞘增厚,束膜形成。结论 再生室中再生过程可分为：（1）液体育孕期（1周内）。（2）纤维连接期（1－2周）。（3）神经连接期（2－4周）。（4）神经成熟期（术后4周以上）。神经再生微环境包括再生条件液、纤维基质带和巨嗜细胞、成纤维细胞、雪旺细胞、内皮细胞等成份。     

Voiding reflex in chronic spinal cord injured cats induced by stimulating and blocking pudendal nerves

AIMS: To induce efficient voiding in chronic spinal cord injured (SCI) cats. METHODS: Voiding reflexes induced by bladder distension or by electrical stimulation and block of pudendal nerves were investigated in chronic SCI cats under alpha-chloralose anesthesia. RESULTS: The voiding efficiency in chronic SCI cats induced by bladder distension was very poor compared to that in spinal intact cats (7.3 +/- 0.9% vs. 93.6 +/- 2.0%, P < 0.05). In chronic SCI cats continuous stimulation of the pudendal nerve on one side at 20 Hz induced large amplitude bladder contractions, but failed to induce voiding. However, continuous pudendal nerve stimulation (20 Hz) combined with high-frequency (10 kHz) distal blockade of the ipsilateral pudendal nerve elicited efficient (73.2 +/- 10.7%) voiding. Blocking the pudendal nerves bilaterally produced voiding efficiency (82.5 +/- 4.8%) comparable to the efficiency during voidings induced by bladder distension in spinal intact cats, indicating that the external urethral sphincter (EUS) contraction was caused not only by direct activation of the pudendal efferent fibers, but also by spinal reflex activation of the EUS through the contralateral pudendal nerve. The maximal bladder pressure and average flow rate induced by stimulation and bilateral pudendal nerve block in chronic SCI cats were also comparable to those in spinal intact cats. CONCLUSIONS: This study shows that after the spinal cord is chronically isolated from the pontine micturition center, bladder distension evokes a transient, inefficient voiding reflex, whereas stimulation of somatic afferent fibers evokes a strong, long duration, spinal bladder reflex that elicits efficient voiding when combined with blockade of somatic efferent fibers in the pudendal nerves.     

Effects of IGF-I gene therapy on the injured rat pudendal nerve

 Injured nerves and their motor units may undergo enhanced recovery when exposed to recombinant human insulin-like growth factor-I (rhIGF-I). The external anal sphincter muscle in the female rat was denervated to model incontinence. The treatment-group muscle was injected with rhIGF-1 plasmid, whereas in the control group the plasmid lacked the cDNA insert and the normal group received neither surgery nor treatment. Electromyography data at 56 days post surgery indicated more reinnervation without fibrillation potentials in the treatment group (2 of 6) than in the control group (0 of 6). The histology of the regenerated axons in the pudendal nerve distal to the crush site also suggested an improved recovery in the treatment group. The number of motor neurons retrogradely labeled with horseradish peroxidase was decreased by 50% following pudendal nerve crush in both experimental groups compared to the normal group. We conclude from these preliminary results that rhIGF-I gene therapy may improve the distal recovery of structure and function. Received: 30 October 2002 / Accepted: 2 September 2002     

Innervation of the levator ani muscles: description of the nerve branches to the pubococcygeus,iliococcygeus, and puborectalis muscles

We described the innervation of the levator ani muscles (LAM) in human female cadavers. Detailed pelvic dissections of the pubococcygeus (PCM), iliococcygeus (ICM), and puborectalis muscles (PRM) were performed on 17 formaldehyde-fixed cadavers. The pudendal nerve and the sacral nerves entering the pelvis were traced thoroughly, and nerve branches innervating the LAM were documented. Histological analysis of nerve branches entering the LAM confirmed myelinated nerve tissue. LAM were innervated by the pudendal nerve branches, perineal nerve, and inferior rectal nerve (IRN) in 15 (88.2%) and 6 (35.3%) cadavers, respectively, and by the direct sacral nerves S3 and/or S4 in 12 cadavers (70.6%). A variant IRN, independent of the pudendal nerve, was found to innervate the LAM in seven (41.2%) cadavers. The PCM and the PRM were both primarily innervated by the pudendal nerve branches in 13 cadavers (76.5%) each. The ICM was primarily innervated by the direct sacral nerves S3 and/or S4 in 11 cadavers (64.7%).     

Interconnections of the upper ventral rami of the human sacral plexus: A reappraisal for dorsal rhizotomy in neurostimulation operations

The extension of a dorsal rhizotomy in bladder stimulation patients is partly determined by connections between the ventral rami of the second, third, and fourth sacral spinal nerves. The literature is inconclusive on interconnections of these ventral rami in the human sacral plexus. The sacral plexuses of ten human cadavers were dissected in this gross anatomy study. In nine cases a branch connecting the ventral rami of the second and third sacral spinal nerves was found. Electron microscopy demonstrated the presence of thick myelinated fibers in this branch. In the male plexuses this branch formed the only link between the second sacral spinal segment and the pelvic plexus. The ventral ramus of the second sacral nerve always contributed to the pudendal nerve, whereas involvement of the ventral rami of the first and third sacral nerves differed individually and intersexually.     

Selective regeneration of motor and sensory axons in an experimental peripheral nerve model without endorgans.

We assessed the selectivity of motor and sensory axon regeneration towards the distal motor and sensory nerve segments that were disconnected from endorgans in a rat silicone Y chamber model. The L5 ventral root was used as a pure motor nerve, and the saphenous nerve was used as a sensory nerve. In experiment 1 (n=11), the proximal stump of the L5 ventral root, a 1-cm-long L5 ventral root segment and a saphenous nerve segment were inserted into a silicone Y chamber. In experiment 2 (n=11), the proximal stump of the saphenous nerve, a L5 ventral root segment and a saphenous nerve segment were inserted into a Y chamber. The distance between the nerve stumps was 5 mm. Six weeks later, the number of regenerated myelinated motor and sensory axons was measured and compared in the distal two channels. Motor axons showed no selective regeneration, but sensory axons did.     

Effects of pudendal nerve injury in the female rat

To test a neurogenic hypothesis for external urethral sphincter (EUS) dysfunction associated with urinary incontinence, the proximal pudendal nerve was crushed in anesthetized retired breeder female rats (n = 5) and compared with a sham lesion group (n = 4). Outcome measures included concentric needle electromyograms (EMGs) from the target EUS, voiding patterns during a 2-hour dark period, and micturition data over a 24-hour period. Fast Blue (FB) was introduced to the crush site at the time of injury and Diamidino Yellow (DY) to the EUS at the time the rats were killed (3 months post-operative), when histological analysis of the nerve and urethra was also performed. EMG records indicated the EUS motor units undergo typical denervation changes followed by regeneration and recovery. Voiding patterns from the crush group show a significant increase of small urine marks in the front third of the cage. At 1-2 weeks post-op, the frequency of voids was significantly increased in the crush group compared to pre-op and late post-op time periods. The mean volume voided in the light phase at the early post-op time was significantly increased in the sham group. Light and electron microscopic patterns seen in nerve and muscle suggest the regenerating motor units maintain a structural integrity. Motoneurons in the lower lumbar cord were labeled with either DY (14. 5 +/- 6.8), FB (31.7 +/- 23.7), or both (35.0 +/- 17.5) tracers, indicating approximately 54% of the crushed pudendal neurons regenerated to the EUS. In conclusion, several measures suggest this reversible crush lesion induces mild urinary incontinence. This animal model is promising for further development of hypotheses regarding neural injury, the pathogenesis of incontinence, and strategies aimed at prevention and treatment. Neurourol. Urodynam. 19:53-69, 2000.     

Histochemical discrimination of fibers in regenerating rat infraorbital nerve.

In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.     

Diffusional delay in local anesthetic block in vitro

The diffusion of lidocaine to myelinated and unmyelinated axons was compared on individual afferent fibers of rabbit vagus nerve. The criterion consisted of the time required for more than 95% completion of the asymptotic increase in impulse conduction time produced by a weak, nonblocking concentration of lidocaine. Measurements on sheathed and desheathed nerves for both myelinated and unmyelinated axons detected an apparent but statistically not significant diffusional lag at the perineurial sheath, averaging four minutes in this model; there was no significant difference in the mean time for attainment of criterion in myelinated and unmyelinated axons, which averaged an additional 13 min in both types of fiber. From these observations the authors conclude that lidocaine diffused as readily through the nodal gap to the excitable membrane of the myelinated fiber as through the Schwann cell mesaxon to the unmyelinated fiber. Thus differential diffusion within a nerve seems unlikely to be a contributing factor to clinical differential block.     

远端神经变性对神经再生放大规律的影响

目的 探讨应用正常供体神经修复预变性受体神经时,再生神经纤维放大的效果.方法 将雄性SD大鼠12只,随机分为变性组、对照组,所有动物均实行手术.变性组应用部分正常腓总神经修复变性8周胫神经远端;对照组应用部分正常腓总神经修复即刻损伤后胫神经远端.术后3个月进行神经电生理测量、肌力测量、组织学观察、神经锇酸染色及有髓神经纤维计数,并计算两组神经再生放大率.结果 变性组和对照组神经传导速度分别为(16.992±3.737)m/s和(23.092±2.788)m/s;肌肉强直收缩力恢复率为(39.642±5.865)%和(71.098±6.778)%;再生有髓神经纤维数分别为1718.2±282.0和3340.0±506.5;神经再生放大率1.581±0.329和3.098± 0.642;以上指标变性组均低于对照组(P＜0.05).结论 应用正常神经修复8周变性神经远端时,神经纤维再生放大率降低,说明损伤远端神经较长时间变性,接受再生神经纤维长入的能力降低.