神经干细胞与雪旺细胞联合应用于神经损伤修复的研究进展

雪旺细胞是周围神经系统特有的神经胶质细胞,也是髓鞘形成细胞,能提供多种细胞因子和营养因子支持受损轴突再生并通过增殖分化形成髓鞘包绕新生轴突。神经干细胞是神经系统发育过程中保留下来的具有多向分化潜能的原始细胞,具有免疫原性低、组织融合性好、可自我更新等特点。雪旺细胞与神经干细胞对于外周神经损伤的修复与再生有着重要作用。近来随着显微外科技术的发展,利用神经导管联合神经干细胞与雪旺细胞桥接神经断端以实现周围神经损伤的修复吸引了越来越多研究者的兴趣。     

Recurrent laryngeal nerve regeneration by tissue engineering

The recurrent laryngeal nerve (RLN) does not regenerate well after it has been cut, and no current surgical methods achieve functional regeneration. Here, we evaluate the functional regeneration of the RLN after reconstruction using a biodegradable nerve conduit or an autologous nerve graft. The nerve conduit was made of a polyglycolic acid (PGA) tube coated with collagen. A 10-mm gap in the resected nerve was bridged by a PGA tube in 6 adult beagle dogs (group 1) and by an autologous nerve graft in 3 dogs (group 2). Fiberscopic observation revealed functional regeneration of the RLN in 4 of the 6 dogs in group 1. No regeneration of the RLN was observed in any dog in group 2. We also tested for axonal transport, and measured the compound muscle action potential. The RLN can be functionally regenerated with a PGA tube, which may act as a scaffold for the growth of regenerating axons.     

Olfactory ensheathing cells in facial nerve regeneration

IntroductionOlfactory ensheathing cell is a unique kind of glia cells, which can promote axon growth. Little is known about the differences between olfactory mucosa olfactory ensheathing cells and olfactory bulb olfactory ensheathing cells in the capability to promote nerve regeneration.ObjectiveTo study the recovery of the rat facial nerve after olfactory ensheathing cells transplantation, and to compare the differences between the facial nerve regeneration of olfactory mucosa-olfactory ensheathing cells and olfactory bulb olfactory bulb olfactory ensheathing cells transplantation.MethodsInstitutional ethical guideline was followed (201510129A). Olfactory mucosa-olfactory ensheathing cells and olfactory bulb olfactory ensheathing cells were cultured and harvested after 7 days in vitro. 36 Sprague Dawley male rats were randomly divided into three different groups depending on the transplanting cells: Group A: olfactory mucosa-olfactory ensheathing cells; Group B: olfactory bulb olfactory ensheathing cells; Group C: DF-12 medium/fetal bovine serum. The main trunk of the facial nerve was transected and both stumps were inserted into a polylactic acid/chitosan conduit, then the transplanted cells were injected into the collagen in the conduits. After 4 and 8 weeks after the transplant, the rats of the three groups were scarified and the facial function score, facial nerve evoked potentials, histology analysis, and fluorescent retrograde tracing were tested and recorded, respectively, to evaluate the facial nerve regeneration and to analysis the differences among the three groups.ResultsOlfactory ensheathing cells can promote the facial nerve regeneration. Compared with olfactory bulb olfactory ensheathing cells, olfactory mucosa olfactory ensheathing cells were more effective in promoting facial nerve regeneration, and this difference was more significant 8 weeks after the transplantation than 4 weeks.ConclusionWe discovered that olfactory ensheathing cells with nerve conduit could improve the facial nerve recovery, and the olfactory mucosa olfactory ensheathing cells are more effective for facial nerve regeneration compared with olfactory bulb olfactory ensheathing cells 8 weeks after the transplantation. These results could cast new light in the therapy of facial nerve defect, and furnish the foundation of auto-transplantation of olfactory mucosa olfactory ensheathing cells in periphery nerve injury.     

Facilitation of facial nerve regeneration using chitosan-β-glycerophosphate-nerve growth factor hydrogel

Conclusion C/GP hydrogel was demonstrated to be an ideal drug delivery vehicle and scaffold in the vein conduit. Combined use autologous vein and NGF continuously delivered by C/GP-NGF hydrogel can improve the recovery of facial nerve defects. Objective This study investigated the effects of chitosan-β-glycerophosphate-nerve growth factor (C/GP-NGF) hydrogel combined with autologous vein conduit on the recovery of damaged facial nerve in a rat model. Methods A 5?mm gap in the buccal branch of a rat facial nerve was reconstructed with an autologous vein. Next, C/GP-NGF hydrogel was injected into the vein conduit. In negative control groups, NGF solution or phosphate-buffered saline (PBS) was injected into the vein conduits, respectively. Autologous implantation was used as a positive control group. Vibrissae movement, electrophysiological assessment, and morphological analysis of regenerated nerves were performed to assess nerve regeneration. Results NGF continuously released from C/GP-NGF hydrogel in vitro. The recovery rate of vibrissae movement and the compound muscle action potentials of regenerated facial nerve in the C/GP-NGF group were similar to those in the Auto group, and significantly better than those in the NGF group. Furthermore, larger regenerated axons and thicker myelin sheaths were obtained in the C/GP-NGF group than those in the NGF group.     

Rabbit facial nerve regeneration in autologous nerve grafts after antecedent injury

OBJECTIVE: The effect of incomplete antecedent injuries on subsequent facial nerve regeneration within cable graft repairs is not known. The purpose of this study is to compare facial nerve regeneration after an immediate and delayed neural cable graft repair. METHOD: Rabbit facial nerve regeneration after complete transectional injuries of the buccal division was compared in two experimental models. In one, a 10-mm segment of the nerve was transected, rotated 180 degrees, and immediately repaired as a cable graft (N=8). In the second, a preliminary nerve crush was allowed to recover over a 4-week period and a 10-mm segment of nerve centered on the crush site was then transected, rotated 180 degrees, and delay repaired as a cable graft (N = 7). Data are presented as total numbers of regenerating myelinated axons that traverse the surgical repair to innervate the cable graft and distal nerve stumps, as well as the percentage of regenerating neurites compared with preoperative pooled and individual controls. Subpopulations of regenerating neurons are delineated to quantify the pattern of neural innervation. RESULTS: Five weeks after cable graft repair both groups had similar myelinated outgrowth from the proximal nerve stump across the proximal anastomosis to innervate the cable graft (3995 +/- 1209 vs. 3284 +/- 651; P = .89). However, the delayed repair group had more intrafascicular regeneration within cable grafts (2261 +/- 931 vs. 1660 +/- 1169; P = .02) and distal nerve stump (1532 +/- 281 vs. 445 +/- 120; P = .004) than the immediate repair group. The immediate repair group had greater extrafascicular nerve regeneration in the cable graft (2335 +/- 1954 vs. 437 +/- 236; P = .001) and more myelin and axonal debris in pre-existing neural fascicles of the cable graft (P = .02) and distal nerve stump (463 +/- 187 vs. 103 +/- 87; P = .02). CONCLUSIONS: Antecedent priming lesions do not enhance axonal survival as determined by regenerating myelinated axonal counts. However, antecedent injuries enhance the efficiency of neural innervation of the affected mimetic musculature by increasing the number of myelinated intrafascicular neural regenerants in the cable graft and distal nerve stump. This is accomplished by two factors: increased perineural fibrosis and decreased intrafascicular myelin and axonal debris.     

改良乳酸-羟基乙酸共聚物神经导管修复猫动眼神经缺损

目的建立一种适合临床使用的神经导管,该导管能修复颅内段动眼神经缺损且方便局部用药,促进神经再生。方法家猫22只随机分成3组:乳酸-羟基乙酸共聚物(PLGA)导管组(A组,10只)、改良PLGA导管组(B组,10只),对照组(C组,2只);右侧动眼神经制成4 mm缺损,A、B两组分别用PLGA导管、改良PLGA导管修复,同时局部给予生理盐水模拟给药,并记录修复神经所需时间;C组不修复。术后定期观察瞳孔对光和眼球运动,术后14周观察再生动眼神经的大体情况,并用光镜、电镜观察,测量神经纤维的数目、直径。结果术后14周时,A、B两组分别有6,7只猫动眼神经功能有一定程度的恢复且神经连续性恢复,光镜及电镜证实神经再生成功,轴突图像分析显示两组平均轴突直径及轴突通过率差异无统计学意义(P>0.05)。C组神经功能及连续性无恢复。结论改良PLGA导管和PLGA导管均能有效修复猫颅内段动眼神经缺损;改良PLGA导管操作更简单,制作材料更容易获得,更接近临床应用,方便局部应用药物促进神经再生。     

Repair of motor nerve gaps with sensory nerve inhibits regeneration in rats

OBJECTIVE: Sensory nerve grafts are often used to reconstruct injured motor nerves, but the consequences of such motor/sensory mismatches are not well studied. Sensory nerves have more diverse fiber distributions than motor nerves and may possess phenotypically distinct Schwann cells. Putative differences in Schwann cell characteristics and pathway architecture may negatively affect the regeneration of motor neurons down sensory pathways. We hypothesized that sensory grafts impair motor target reinnervation, thereby contributing to suboptimal outcomes. This study investigated the effect of motor versus sensory grafts on nerve regeneration and functional recovery. STUDY DESIGN: The authors conducted a prospective, randomized, controlled animal study. METHODS: Fifty-six Lewis rats were randomized to seven groups of eight animals each. Five-millimeter tibial nerve defects were reconstructed with motor or sensory nerve grafts comprised of single, double, triple, or quadruple cables. Tibial nerve autografts served as positive controls. Three weeks after reconstruction, nerves were harvested for histologic examination and quantitative histomorphometric analysis. Wet muscle masses provided an index of functional recovery. RESULTS: Nerve regeneration was significantly greater across motor versus sensory nerve grafts independent of graft cross-sectional area or cable number. Motor grafts demonstrated increased nerve density, percent nerve, and total fiber number (P < .05). Normalized wet muscle masses trended toward improved recovery in motor versus sensory groups. CONCLUSIONS: Reconstruction of tibial nerve defects with nerve grafts of motor versus sensory origin enhanced nerve regeneration independent of cable number in a rodent model. Preferential nerve regeneration through motor nerve grafts may also promote functional recovery with potential implications for clinical nerve reconstruction.     

Experimental study on repair of the facial nerve with Schwann cells transfected with GDNF genes and PLGA conduits

Conclusions. Schwann cells transfected by GDNF genes+PLGA were superior to Schwann cells+PLGA and direct anastomesis. This is a new and effective strategy for repair of facial nerve defects. Objective. To evaluate the effect of bioactive artificial nerve conduits in the repair of facial nerve defects in Sprague-Dawley rats. Materials and methods. Schwann cells were harvested and transfected with PcDNA3.1(+)/GDNF. After injection with Schwann cells, the conduits were cultured in the culture medium for 2 weeks. Thirty female Sprague-Dawley rats were selected and randomly divided into three groups (A, B, and C), which were treated as follows: A, direct anastomesis; B, Schwann cells+PLGA conduits; C, Schwann cells transfected by GDNF genes+PLGA conduits. General observation, electrophysiological study, histological study, and image analysis were performed 2 weeks, 1 month, 2 months, and 3 months postoperatively. Results. The recovery of nerve regeneration and electrophysiological results in group C were superior to those in groups A and B; the difference was statistically significant (p<0.01).     

Laryngeal abductor muscle reinnervation in a pig model

OBJECTIVE: To develop a large animal model for studies of laryngeal abductor reinnervation. MATERIAL AND METHODS: Six minipigs underwent unilateral anastomosis of the phrenic nerve-abductor branch of the recurrent laryngeal nerve (RLN). Polyhydroxybutyrate (PHB) conduits were used for repair. At each of 30, 60 and 120 days, 2 animals underwent video laryngeal endoscopy (VLE) and were then killed. VLE was also performed in the 120-day pair at 60 days. Nerve-conduit-nerve-muscle samples were fixed for light and immunofluorescence (pan-neurofilaments, S-100) microscopy. Laryngeal muscles were harvested (myosin heavy chain analysis). RESULTS: VLE showed recovery of abductor function in 1 animal at 60 days and in 1 at 120 days. Haematoxylin-eosin staining demonstrated a complex inflammatory response. Eosinophil recruitment was observed. Stepwise regeneration and reorganization of the distal nerve between 30 and 120 days was observed with pan-NF staining. The mean minimum diameter in the reinnervated posterior crico-arytenoids tended to increase for up to 120 days. CONCLUSIONS: Anastomosis of the phrenic nerve-abductor branch of the RLN with a PHB conduit in a pig can result in functional and histological recovery within 2-4 months and appears to at least sustain abductor muscle fibre morphology. Recovery occurs despite a complex inflammatory response, which may be an essential part of healing rather than inhibitory.     

Nerve regeneration in the peripheral nervous system versus the central nervous system and the relevance to speech and hearing after nerve injuries

Schwann cells normally form myelin sheaths around axons in the peripheral nervous system (PNS) and support nerve regeneration after nerve injury. In contrast, nerve regeneration in the central nervous system (CNS) is not supported by the myelinating cells known as oligodendrocytes. We have found that: 1) low frequency electrical stimulation can be used to elevate cAMP thereby promoting regeneration of CNS axons and 2) a conditioning lesion, created by a crush of the peripheral branch of the dorsal root ganglion sensory neurons along with a simultaneous cut of these axons in the CNS, promotes even greater neural outgrowth than electrical stimulation. The effectiveness of the lesion results from both an acceleration of axon outgrowth and an increase in the rate of axon growth. However, electrical stimulation remains a more viable treatment of nerve injuries to stimulate regeneration and has been successfully used to promote development of the auditory pathways in children with severe to profound deafness who use cochlear implants. Without nerve regeneration, there is only a random reinnervation of affected muscles. An example occurs when the laryngeal nerve attempts to reinnervate the vocal cords after injury, causing deficits in speech. Synkinesis occurs when reinnervation of antagonistic muscles effectively paralyze the vocal cords and, in turn, severely compromises speech. The misdirection of laryngeal nerve reinnervation can be alleviated surgically by strategies favoring inspiratory abduction.Learning outcomesReaders of this article will gain an understanding of (1) the potential for axon regeneration in the central nervous system and (2) problems and possible solutions for random reinnervation of laryngeal muscles for speech.     

Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig

The presence and localization of different neuropeptides and other putative neurotransmitters or -modulators were examined by immunohistochemistry in the cochleovestibular end organs and in neurons innervating them in rats and guinea pigs. In the organ of Corti neural elements beneath inner hair cells showed immunoreactivity for enkephalin (ENK), calcitonin gene-related peptide (CGRP), L-glutamate decarboxylase (GAD), substance P (SP) and tyrosine hydroxylase (TH). Nerve chalices of type I vestibular hair cells contained SP and GAD, but not consistently. SP was only occasionally observed in neuronal cell bodies of the 8th cranial nerve but fine fibers with different neuroactive substances were seen in the nerve trunk in the following relative numbers: TH greater than SP greater than CGRP greater than ENK. The present data demonstrate the presence of several different neuroactive substances in the rat and guinea pig inner ear suggesting a multiplicity of neurotransmitters or -modulators in this system.     

Preliminary study of the facial nerve regeneration in the chamber: the influence of myelin basic protein]

OBJECTIVE: To study the role of exogenous myelin basic protein(MBP) in neural repairment. METHODS: Adult New Zealand rabbits were employed in vivo preparation. A 12 microL nerve growth chamber was created by suturing the proximal and distal stumps of a transected facial never (FN) trunk into a tube. The regenerated nerves within the chambers were dissected and fixed for histological studies with light microscope at 4, 6 and 8 weeks respectively following the surgery. RESULTS: Morphological analysis of nerves showed no difference between the MBP and control group in the size of the regeneration FN within the chambers, diameters of myelinated axons, thickness of myelin sheath and number of myelin axons grew into the distal end of chamber at 4 weeks. At 6 and 8 weeks after operation, the MBP group showed a more mature-appearance regenerative nerve comparing to control group. Especially, the enhancement of maturation in the regeneration axons was very noticeable at 6 weeks. CONCLUSION: The study showed that pharmacological administration of exogenous MBP within a chamber at the time of entubational nerve repair enhances regeneration of myelinated axons across the sectioned ends of FN.     

微波变性自体骨骼肌和异体神经修复面神经缺损的实验研究

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Rabbit facial nerve regeneration in NGF-containing silastic tubes

Previous reports suggest that exogenous nerve growth factor (NGF) enhanced nerve regeneration in rabbit facial nerves.¹ Rabbit facial nerve regeneration in 10-mm Silastic® tubes prefilled with NGF was compared to cytochrome C (Cyt. C), bridging an 8-mm nerve gap. Three weeks following implantation, NGF-treated regenerates exhibited a more mature fascicular organization and more extensive neovascularization than cytochrome-C-treated controls. Morphometric analysis at the midtube of 3- and 5-week regenerates revealed no significant difference in the mean number of myelinated or unmyelinated axons between NGF- and cytochrome-C-treated implants. However, when the number of myelinated fibers in 5-week regenerates were compared to their respective preoperative controls, NGF-treated regenerates had recovered a significantly greater percentage of myelinated axons than cytochrome-C-treated implants (46% vs. 18%, respectively). In addition, NGF-containing chambers reinnervated a higher percentage of myelinated axons in the distal transected neural stumps (49% vs. 34%). Behavioral and electrophysiologic studies demonstrated spontaneous and induced activities in the target muscles when approximately one third of the myelinated axons were recovered in the midchamber (1280 axons). Horseradish peroxidase (HRP) studies demonstrated retrograde axonal transport to the midchamber and proximal transected neural stump. PC12 bioassay demonstrated persistent NGF activity in the intrachamber fluids at 3 (5:1 dilution) and 5 (2:1 dilution) weeks of entubation. Electrophysiologic tests demonstrated a slow conduction velocity of a propagated electrical impulse (43.5 m/s^?1 vs. 67 m/s^?1) and shallow wide compound action potential. In wider defects (15-mm chambers) and longer entubation periods (7 weeks), no regeneration or NGF activity was seen. Therefore, exogenous NGF provides an early but limited neurotrophic effect on the regeneration of the rabbit buccal division of the facial nerve and a limited behavioral and physiological improvement in the target muscles.     

The use of muscle autograft or nerve allograft denatured by microwave for repair of gaps in rat facial nerve]

OBJECTIVE: Attempt to enhance recovery of rat facial nerve through different grafts. METHODS: Rats facial nerve mandible branch gap was repaired using different grafts, either muscle autograft or sural nerve allograft denatured by microwave. Axonal regeneration was studied in 10th week after insertion of the denatured muscle autografts or sural nerve allografts and compared with results found in autologous sural nerve grafts used as controls. Axonal regeneration, Schwann cell behavior and efficacy of nerve and muscle were quantified using CB-HRP retrograde trace, HE staining, Flamming staining and electromyography. RESULTS: Denatured nerve allografts and muscle autografts supported the higher rates and volumes of axonal regeneration. Nerve allografts had the higher degree of myelin sheath developing. CONCLUSION: Nerve autograft and muscle autograft denatured by microwave are convenient, source sufficient, higher efficient grafts for repairing facial nerve gap and have potential clinical use.     

面神经修复中再生室内髓鞘碱性蛋白的作用初探

目的探讨外源性髓鞘碱性蛋白（myelinbasicprotein,MBP）在家兔面神经再生室修复中的作用。方法将33只家兔横断的面神经干近、远端缝于硅胶管壁上,形成约12μL大小的神经再生室。一侧为实验组,将MBP注入再生室内;对侧为对照组不注任何物质。分别在术后4、6、8周处死动物,切取标本,在光镜下行组织形态学观察。结果形态学分析表明术后4周2组再生室内再生面神经的有髓轴突直径、髓鞘厚度及长入再生室远端有髓轴突数差异无显著性(P>0.05),随着时间的延长(术后6、8周),MBP组较对照组再生面神经显得更为成熟,6周时再生轴突成熟程度差异更明显。结论MBP有促进家兔面神经再生修复的作用,但在活体内增强受损轴突生长的机理以及MBP促进FN轴突再生的作用时间尚不清楚。     

RNA干扰神经干细胞中NgR基因的表达修复大鼠面神经缺损的体外实验研究

目的：应用RNA干扰技术抑制神经干细胞中NgR基因表达,观察神经干细胞的体外分化,为体内面神经修复提供营养支持。方法：采用PCR扩增,限制性内切酶酶切,T4DNA连接酶连接,将NgR与pGCsi载体连接,构建重组载体NgR shRNA,通过测序鉴定。NgR shRNA通过Lipofectamine 2000感染神经干细胞,荧光显微镜下观察EGFP的表达,WesternBlot检测NgR的表达,免疫细胞化学鉴定转染前后神经干细胞向神经元分化的比例,转染后的神经干细胞种植于PLGA管,扫描电镜观察。结果：成功构建NgR shRNA质粒,成功感染神经干细胞,Western Blot结果表明干扰后的神经干细胞中的NgR表达减弱、免疫细胞化学结果示干扰后的神经干细胞向神经元分化比例明显高于对照组（P＜0．01）,差异有统计学意义。结论：成功构建NgR shRNA质粒,感染神经干细胞,使神经干细胞向神经元进一步分化,能更有效促进神经轴突的再生,为面神经损伤修复提供高效稳定的基因平台。     

Facial nerve regeneration through semipermeable chambers in the rabbit.

Peripheral neural regeneration, over a 10-mm transectional gap, was determined in 70 rabbit buccal divisions of the facial nerve using two entubational systems (semipermeable and impermeable silicone chambers) prefilled with three natural occurring media (serum, blood, and saline) during a 5-week period. The number of myelinated axonal regenerates at the midchamber and at 2 mm in the distal transected neural stump were counted in each group and compared to pooled myelinated axonal counts in 9 normal rabbit buccal divisions of the facial nerve. Semipermeable porous chambers had an overall greater regeneration success rate (75% vs. 42.8%) and regained, on the average, a higher number of myelinated axons (51.4% vs. 26.1%) than silicone chamber regenerates. Semipermeable chambers prefilled with serum or blood had significantly higher regeneration success rates, myelinated axonal counts, and percentages of neural innervation of the distal transected neural stump. Both entubational systems produced similar axonal counts with intraluminal saline. The highest overall success rate (93.7%) and average number of myelinated axons per chamber (3072) were achieved in semipermeable chambers prefilled with serum. The greatest variability in myelinated axonal counts (0 to 3266 axons) and percentage of distal stump innervation (5.5% to 98.1%) was seen in silicone chambers filled with saline. The percentage of myelinated axons from the midchamber that innervated the distal stump was greater in semipermeable chambers with blood (73%) and serum (54%) than in silicone saline chambers (43%). On the average, the distal stumps from semipermeable chambers filled with serum (47%) and blood (33.5%) regained a higher percentage of normal myelinated axonal counts than silicone-saline chambers (12.5%). These results suggest that both the construction of entubational chamber and the intraluminal medium can have significant influence on neurite regeneration. Semipermeable chambers prefilled with serum have a strong neurite-promoting potential in peripheral neural regeneration of rabbit facial nerves.     

Effects of external beam radiation in the rat tibial nerve after crush, transection and repair, or nerve isograft paradigms

INTRODUCTION: In head and neck surgery, radiation therapy is often administered to an injured nerve. Previous studies have examined the effects of either preoperative or postoperative radiation on nerve regeneration in rodents. In these studies, histomorphometric analysis was performed up to 8 month postoperatively. Given the exceptional neuroregenerative capacity of rodents, significant differences in nerve regeneration may go undetected if nerves are evaluated at such distant postoperative time points. This study is designed with a more appropriate model and investigates the effects of radiation after three common nerve injury paradigms. METHODS: Sixty-four Lewis rates were randomized to 8 groups corresponding to uninjured, tibial nerve crush, transection and repair, or reconstruction with isografts. Half of the animals in each of these paradigms (n = 8 per group) were treated with 10 Gy of external beam radiation to the site of nerve injury at 7 days postoperatively. On postoperative day 28, functional recovery and histomorphometric assessment was performed. RESULTS: For a given paradigm of nerve injury, no significant differences in nerve fiber number, neural density, neural debris, or fiber width were noted between the control and radiated groups, and radiation did not affect functional recovery. CONCLUSION: Radiation had no discernible effect on nerve regeneration or functional recovery in the rodent nerve injury models studied. All assessments were made at time points suitable for detecting differences in nerve regeneration between groups. These findings suggest that administration of radiation to fields containing injured peripheral nerve is unlikely to adversely affect functional outcomes.     

Morphological relationships of peptidergic and noradrenergic nerve terminals to olivocochlear neurones in the rat

In the rat, the outer hair cells in the cochlea receive direct synaptic input from neurones in the ventral nucleus of the trapezoid body. These so-called medial olivocochlear neurones exert an inhibitory influence on the cochlear neural output. Electrophysiological in vitro studies suggest that the activity of medial olivocochlear neurones may be affected by a variety of neuropeptides as well as noradrenaline, but anatomical confirmation of direct synaptic input is still lacking. We have investigated, at the light microscopical level, the morphological relationships between terminals containing noradrenaline, substance P, cholecystokinin and leu-enkephalin, and medial olivocochlear neurones in the rat. A retrograde tracer was injected into the cochlea to label medial olivocochlear neurones and a double labelling immunocytochemical method was used to visualise the retrograde tracer as well as the neurotransmitters within each brain section. Light microscopical analysis revealed nerve endings containing substance P, cholecystokinin and leu-enkephalin in close apposition to the dendrites of medial olivocochlear neurones, and nerve endings containing dopamine-beta-hydroxylase, a marker for noradrenaline, in close contact with the somata as well as dendrites of medial olivocochlear neurones. Although the technique cannot prove the existence of functional synaptic contacts, the results are broadly consistent with electrophysiological data and suggest a direct input to medial olivocochlear neurones from substance P, cholecystokinin, leu-enkephalin and noradrenaline-containing neural pathways. Differences in the densities and spatial distribution of the various neuropharmacological inputs suggest differences in the relative strengths and possible roles of these diverse inputs to the olivocochlear system.