Effect of locally delivered IGF‐1 on nerve regeneration during aging: An experimental study in rats

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin‐like growth factor 1 (IGF‐1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF‐1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF‐1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF‐1 significantly improved axon number, diameter, and density. IGF‐1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF‐1 treatment. Muscle Nerve, 2009     

Agmatine promotes expression of brain-derived neurotrophic factor in brainstem facial nucleus in the rat facial nerve injury model★

BACKGROUND: Studies have shown that agmatine can reduce inhibition of neuronal regeneration by in-creasing cyclic adenosine monophosphate and brain-derived neurotrophic factor (BDNF) in the hippocampus of morphine-dependent rats. The hypothesis that agmatine exerts similar effects on facial nerve injury de-serves further analysis. OBJECTIVE: To study the effects of peritoneal agmatine injection on BDNF levels in the rat brainstem af-ter facial nerve injury. DESIGN, TIME AND SETTING: A controlled animal experiment was performed at the Department of Otolaryngology-Head and Neck Surgery at the Second Affiliated Hospital, Chongqing University of Medical Sciences (Chongqing, China), between October and December in 2007. MATERIALS: Twenty-four male Sprague-Dawley rats were randomly divided into a control, a lesion, and an agmatine treatment group, with eight rats in each group. Bilateral facial nerve anastomosis was induced in the lesion and agmatine treatment groups, while the control group remained untreated. A rat BDNF En-zyme-linked immunosorbent assay kit was used to measure BDNF levels in the brainstem facial nucleus. METHODS: Starting on the day of lesion, the agmatine group received a peritoneal injection of 100 mg/kg agmatine, once per day, for a week, whereas rats in the lesion group received saline injections. MAIN OUTCOME MEASURES: BDNF levels in the brainstem containing facial nucleus were measured by ELISA. RESULTS: Twenty-four rats were included in the final analysis without any loss. Two weeks after lesion, BDNF levels were significantly higher in the lesion group than in the control group (P < 0.01). A significant increase was noted in the agmatine group compared to the lesion group (P < 0.01). CONCLUSION: Agmatine can substantially increase BDNF levels in the rat brainstem after facial nerve injury. Key Words: agmatine; facial nerve injury; brain-derived neurotrophic factor; ELISA     

外源性神经生长因子诱导下自体静脉桥接修复神经缺损

背景：采用自体神经游离移植修复神经缺损效果比较理想,但有其弊端。为此寻求一种更佳修复神经缺损的治疗方法。 目的：验证及外源性神经生长因子诱导下自体静脉桥接神经缺损对神经再生的影响。 方法：采用Wistar大鼠建立周围神经缺损模型。随机将大鼠分为3组。实验组采用自体静脉桥接并注入神经生长因子;对照组采用自体静脉桥接并注入生理盐水;标准组采用自体神经桥接。分别于术后1,3个月,对实验动物进行活体观察,电生理检测及组织学检测。 结果与结论：3组实验动物均有神经再生及修复表现,但程度不同。实验组失神经表现恢复的较对照组早,电生理检测运动神经传导速度快,组织学检查再生神经纤维数量及质量明显高于对照组(P < 0.05);与“金标准”的自体神经桥接组比较无显著性意义(P > 0.05)。结果提示采用自体静脉桥接+神经生长因子诱导对周围神经缺损后的再生、修复具有有促进作用,可以使再生神经纤维的数量增加并显著提高再生神经纤维质量。     

Clinical applications of peripheral nerve allotransplantation. Present medical experience and review of the literature

Nerve autograft has become recently the gold standard for the reconstruction of surgical nerve gap. Functional results of using this technique continue to improve with the advances in micro-neurosurgery and with greater understanding of neurobiology and nerve regeneration. Surgical reconstruction of extensive nerve injuries frequently exhausts the patient's own source of expandable autogenous nerve grafts. Nerve allografts would offer a limitless supply of graft material. The requirement of only transient immunosuppression to support the regeneration of host axons across the nerve allograft toward distal host receptors renders the nerve allograft an advantageous option for these otherwise irreparable nerve injuries. The methods of graft harvesting, preservation and patients' immunosuppressive regimen as well as the clinical outcomes following the nerve allotransplantation are discussed.     

Agmatine promotes expression of brain-derived neurotrophic factor in brainstem facial nucleus in the rat facial nerve injury model★

BACKGROUND: Studies have shown that agmatine can reduce inhibition of neuronal regeneration by increasing cyclic adenosine monophosphate and brain-derived neurotrophic factor (BDNF) in the hippocampus of morphine-dependent rats. The hypothesis that agmatine exerts similar effects on facial nerve injury deserves further analysis.OBJECTIVE: To study the effects of peritoneal agmatine injection on BDNF levels in the rat brainstem after facial nerve injury.DESIGN, TIME AND SETTING: A controlled animal experiment was performed at the Department of Otolaryngology-Head and Neck Surgery at the Second Affiliated Hospital, Chongqing University of Medical Sciences (Chongqing, China), between October and December in 2007.MATERIALS: Twenty-four male Sprague-Dawley rats were randomly divided into a control, a lesion, and an agmatine treatment group, with eight rats in each group. Bilateral facial nerve anastomosis was induced in the lesion and agmatine treatment groups, while the control group remained untreated. A rat BDNF Enzyme-linked immunosorbent assay kit was used to measure BDNF levels in the brainstem facial nucleus.METHODS: Starting on the day of lesion, the agmatine group received a peritoneal injection of 100 mg/kg agmatine, once per day, for a week, whereas rats in the lesion group received saline injections.MAIN OUTCOME MEASURES: BDNF levels in the brainstem containing facial nucleus were measured by ELISA.RESULTS: Twenty-four rats were included in the final analysis without any loss. Two weeks after lesion, BDNF levels were significantly higher in the lesion group than in the control group (P<0.01). A significant increase was noted in the agmatine group compared to the lesion group (P<0.01).CONCLUSION: Agmatine can substantially increase BDNF levels in the rat brainstem after facial nerve injury.     

Effects of motor and sensory nerve transplants on amount and specificity of sciatic nerve regeneration

Nerve regeneration after complete transection does not allow for adequate functional recovery mainly because of lack of selectivity of target reinnervation. We assessed if transplanting a nerve segment from either motor or sensory origin may improve specifically the accuracy of sensory and motor reinnervation. For this purpose, the rat sciatic nerve was transected and repaired with a silicone guide containing a predegenerated segment of ventral root (VR) or dorsal root (DR), compared to a silicone guide filled with saline. Nerve regeneration and reinnervation was assessed during 3 months by electrophysiologic and functional tests, and by nerve morphology and immunohistochemistry against choline acetyltransferase (ChAT) for labeling motor axons. Functional tests showed that reinnervation was successful in all the rats. However, the two groups with a root allotransplant reached higher degrees of reinnervation in comparison with the control group. Group VR showed the highest reinnervation of muscle targets, whereas Group DR had higher levels of sensory reinnervation than VR and saline groups. The total number of regenerated myelinated fibers was similar in the three groups, but the number of ChAT+ fibers was slightly lower in the VR group in comparison with DR and saline groups. These results indicate that a predegenerated root nerve allotransplant enhances axonal regeneration, leading to faster and higher levels of functional recovery. Although there is not clear preferential reinnervation, regeneration of motor axons is promoted at early times by a motor graft, whereas reinnervation of sensory pathways is increased by a sensory graft.     

Permanent rescue of lesioned neonatal motoneurons and enhanced axonal regeneration by adenovirus-mediated expression of glial cell-line-derived neurotrophic factor

Axotomy of peripheral nerves in neonatal rats induces motoneuron death that can be delayed but not arrested by the application of several neurotrophic factors (NFs) or adenoviral vectors carrying genes for NFs. We tested whether an adenoviral vector carrying the gene for glial cell-line-derived neurotrophic factor (Adv.RSV-GDNF) would prevent neonatal motoneuron death after facial nerve transection or crush. Nerve transection eliminates the pathway for axonal regeneration, while nerve crush preserves the pathway necessary for target reinnervation that may be required for the permanent rescue of motoneurons. Both types of injury cause substantial motoneuron death in neonatal animals. Adv.RSV-GDNF or a control vector carrying the β-galactosidase gene (Adv.RSV-βgal) was injected into facial muscles 2 days before the nerve was transected, or Adv.RSV-GDNF, Adv.RSV-βgal, Adv.dl312 (a vector lacking a transgene), or vehicle was injected into facial muscles immediately after nerve crush. Four weeks after nerve transection, few motoneurons survived after Adv.RSV-GDNF and Adv.RSV-βgal treatment (6.1% and 2.4%, respectively). Four weeks after nerve crush, 40% of the motoneurons survived after Adv.RSV-GDNF treatment but only 17% survived in control groups. By 20 weeks, 39% of the motoneurons of the Adv.RSV-GDNF treatment groups survived but only 15–19% survived in controls. The numbers of myelinated axons of the buccal nerve branch of Adv.RSV-GDNF treatment groups were also higher than controls at 4 and 20 weeks (24% and 100% compared to 4.4–6.2% and 25–33% for Adv.RSV-GDNF and controls, respectively). By 20 weeks, Adv.RSV-GDNF-treated animals recovered 50% of the contralateral vibrissal function, while in controls only 5–11% of function was restored. J. Neurosci. Res. 54:766–777, 1998. © 1998 Wiley-Liss, Inc.     

Calcitonin pump improves nerve regeneration after transection injury and repair

Introduction: After nerve injury, excessive calcium impedes nerve regeneration. We previously showed that calcitonin improved nerve regeneration in crush injury. We aimed to validate the direct effect of calcitonin on transected and repaired nerve. Methods: Two rat groups (n = 8) underwent sciatic nerve transection followed by direct repair. In the calcitonin group, a calcitonin‐filled mini‐osmotic pump was implanted subcutaneously, with a catheter parallel to the repaired nerve. The control group underwent repair only, without a pump. Evaluation and comparison between the groups included: (1) compound muscle action potential recording of the extensor digitorum longus (EDL) muscle; (2) tetanic muscle force test of EDL; (3) nerve calcium concentration; and (4) nerve fiber count and calcified spot count. Results: The calcitonin pump group showed superior recovery. Conclusions: Calcitonin affects injured and repaired peripheral nerve directly. The calcitonin‐filled mini‐osmotic pump improved nerve functional recovery by accelerating calcium absorption from the repaired nerve. This finding has potential clinical applications. Muscle Nerve 51 : 229–234, 2015     

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.     

Comparison of continuous and discontinuous FK506 administration on autograft or allograft repair of sciatic nerve resection

An immunosuppressant drug that also possesses neuroregenerative properties, FK506 enhances the rate of axonal regeneration and improves recovery after nerve lesions. Nevertheless, prolonged immunosuppression may not be justified to assure the success of nerve regeneration. In this study, we compare the effects of continuous and discontinuous FK506 treatment on regeneration and reinnervation after sciatic nerve resection repaired with autologous or allogenic grafts in the mouse. For each type of repair, one group received FK506 (5 mg/kg) for 4 months, whereas a second group was treated with FK506 at 5 mg/kg for 5 weeks followed by 3 mg/kg for 4 weeks; a control group received saline only. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hind paw over 4 months after operation. Morphological analysis of the regenerated nerves was performed at the termination of the study. Autografts and allografts treated with sustained FK506 (5 mg/kg) reached high levels of reinnervation and followed a course of recovery faster than controls. The numbers of myelinated fibers also were similar. Allografts without immunosuppression demonstrated a slower rate of regeneration, exhibiting lower final levels of recovery compared with other groups and containing fewer numbers of regenerating myelinated fibers. Withdrawal of immunosuppressant therapy resulted in a decline in the degree of reinnervation in all functions tested during the third month, with stabilization between the third and fourth months. The number of regenerated myelinated fibers in the group was significantly lower than in autografts. Thus, continuous or discontinuous FK506 administration slightly accelerated the rate of reinnervation in autografts. In allograft repair, FK506 significantly enhanced both the rate and degree of regeneration and recovery, but its withdrawal resulted in graft rejection, a marked deterioration in function, and loss of regenerating fibers.     

Olfactory ensheathing cells promote nerve regeneration and functional recovery after facial nerve defects

Olfactory ensheathing cells from the olfactory bulb and olfactory mucosa have been found to increase axonal sprouting and pathfinding and promote the recovery of vibrissae motor performance in facial nerve transection injured rats. However, it is not yet clear whether olfactory ensheathing cells promote the reparation of facial nerve defects in rats. In this study, a collagen sponge and silicone tube neural conduit was implanted into the 6-mm defect of the buccal branch of the facial nerve in adult rats. Olfactory ensheathing cells isolated from the olfactory bulb of newborn Sprague-Dawley rats were injected into the neural conduits connecting the ends of the broken nerves, the morphology and function of the regenerated nerves were compared between the rats implanted with olfactory ensheathing cells with the rats injected with saline. Facial paralysis was assessed. Nerve electrography was used to measure facial nerve-induced action potentials. Visual inspection, anatomical microscopy and hematoxylin-eosin staining were used to assess the histomorphology around the transplanted neural conduit and the morphology of the regenerated nerve. Using fluorogold retrograde tracing, toluidine blue staining and lead uranyl acetate staining, we also measured the number of neurons in the anterior exterior lateral facial nerve motor nucleus, the number of myelinated nerve fibers, and nerve fiber diameter and myelin sheath thickness, respectively. After surgery, olfactory ensheathing cells decreased facial paralysis and the latency of the facial nerve-induced action potentials. There were no differences in the general morphology of the regenerating nerves between the rats implanted with olfactory ensheathing cells and the rats injected with saline. Between-group results showed that olfactory ensheathing cell treatment increased the number of regenerated neurons, improved nerve fiber morphology, and increased the number of myelinated nerve fibers, nerve fiber diameter, and myelin sheath thickness. In conclusion, implantation of olfactory ensheathing cells can promote regeneration and functional recovery after facial nerve damage in rats.     

Acceleration of peripheral nerve regeneration following FK506 administration

Purpose: The severe functional and sensory deficits seen following injury to peripheral nerves makes facilitation of nerve regeneration a primary goal of the reconstructive surgeon. This study examines whether daily administration of FK506 or Cyclosporin A expedites peripheral nerve regeneration following neurotmetic injury in a rat model Methods: Inbred Buffalo rats were randomized to three experimental groups. Group I rats served as untreated controls. Rats in groups II and III received daily subcutaneous CsA (5 mg/kg), and FK506 (1 mg/kg), respectively. Each animal underwent unilateral posterior tibial nerve transection with immediate epineurial reapproximation. Functional recovery of the injured limb was assessed by serial walking track analysis. Nerve regeneration was assessed histomorphometrically via light microscopy. Results: Return of hindlimb function in control animals occurred at 32 days post injury. CsA and FK506-treated transection animals recovered at 26 and 18 days post injury, respectively. Statistically significant greater fiber density and percent neural tissue were seen in FK506- treated animals compared to control animals four weeks post transection. Conclusions: This data suggest that the daily systemic administration of both CsA and FK506 accelerate the rate of functional regeneration, following neurotmetic injuries in tbc rat model. FK506's effect on nerve growth is significantly greater than that of CsA.     

Autogenous inside-out versus standard vein and skeletal muscle-combined grafting for facial nerve repair

BACKGROUND: In the repair of nerve defects, collapse of the venous wall, as a result of vein grafting alone, could impede nerve regeneration. Therefore, vein lumens filled with muscle and nerve segments have been used to bridge nerve defects. OBJECTIVE: To compare the effects of autogenous, inside-out, vein-skeletal, muscle-combined grafting versus standard, vein-skeletal, muscle-combined grafting for the repair of facial nerve defects. DESIGN, TIME AND SETTING: A randomized, controlled, neuroanatomical, animal study was erformed at the Animal Experimental Center and Laboratories of the Capital Medical University Xuanwu Hospital and the Peking Union Medical College Hospital from September 2007 to October 2008. MATERIALS: A total of 10 healthy, male, New Zealand rabbits, aged 6 months, were randomly assigned to inside-out, vein-skeletal, muscle-combined grafting and standard, vein-skeletal, muscle-combined grafting groups, with 5 rabbits in each group. METHODS: A 20-mm gap in the buccal branch of the right facial nerve was made in each animal, which was respectively repaired with inside-out, vein-skeletal, muscle-combined grafts or standard vein-skeletal muscle-combined grafts. MAIN OUTCOME MEASURES: At 6 months after implantation, evoked maximal compound muscle action potentials were recorded on bilateral facial nerves using electromyogram. Myelinated nerve fibers of the regenerating nerves were quantified using myelin sheath osmic acid staining. RESULTS: There was no significant difference between the groups in terms of ratios of bilateral amplitude and latency of compound muscle action potential (P> 0.05). Moreover, morphology of regenerating nerves and quantity of myelinated nerve fibers were similar between the groups (P > 0.05). CONCLUTION: Compared with standard vein grafting, the inside-out vein grafting did not significantly improve nerve regeneration. Therefore, it is not necessary to utilize inside-out vein grafting for the repair of nerve defects, in particular with the combined use of autogenous vein and skeletal muscle grafts.     

FK506 increases peripheral nerve regeneration after chronic axotomy but not after chronic schwann cell denervation

Poor functional recovery after peripheral nerve injury is attributable, at least in part, to chronic motoneuron axotomy and chronic Schwann cell (SC) denervation. While FK506 has been shown to accelerate the rate of nerve regeneration following a sciatic nerve crush or immediate nerve repair, for clinical application, it is important to determine whether the drug is effective after chronic nerve injuries. Two models were employed in the same adult rats using cross-sutures: chronic axotomy and chronic denervation of SCs. For chronic axotomy, a chronically (2 months) injured proximal tibial (TIB) was sutured to a freshly cut common peroneal (CP) nerve. For chronic denervation, a chronically (2 months) injured distal CP nerve was sutured to a freshly cut TIB nerve. Rats were given subcutaneous injections of FK506 or saline (5 mg/kg/day) for 3 weeks. In the chronic axotomy model, FK506 doubled the number of regenerated motoneurons identified by retrograde labeling (from 205 to 414 TIB motoneurons) and increased the numbers of myelinated axons (from 57 to 93 per 1000 microm2) and their myelin sheath thicknesses (from 0.42 to 0.78 microm) in the distal nerve stump. In contrast, after chronic denervation, FK506 did not improve the reduced capacity of SCs to support axonal regeneration. Taken together, the results suggest that FK506 acts directly on the neuron (as opposed to the denervated distal nerve stump) to accelerate and promote axonal regeneration of neurons whose regenerative capacity is significantly reduced by chronic axotomy.     

Long-term observation on the changes of somatotopy in the facial nucleus after nerve suture in the cat: Morphological studies using retrograde labeling

To examine the time course of plasticity of the cranial nucleus during axonal regeneration, we followed the topographical reorganization of the cat facial nucleus (FN) up to 24 months after facio-facial nerve suture using retrograde labeling methods. The trunk of the temporal-zygomatico-orbital and both superior and inferior buccolabial branches (defined as main branch) of the facial nerve was cut and sutured again under ketamine hydrochloride anesthesia. At 11-722 days after nerve suture, Fast Blue (FB) and 1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (Dil) or horseradish peroxidase (HRP) were injected into the distal part of the sutured main branch and the unoperated posterior auricular branch, respectively. Until about 3 months after suture, the topographical pattern in FN was similar to that observed in normal cats. At about 4 months after suture, FB-labeled motoneurons were distributed not only in the lateral part (including intermediate, dorsal and ventrolateral divisions) but also in the medial subdivision of FN. After a survival period of 18-24 months, FB-labeled neurons were found all over the FN, and their number increased significantly. Interestingly, in the longer survival cases, we noticed that the Dil- or HRP-labeled posterior auricular branch motoneurons also showed a tendency to distribute outside the medial region. The present study showed that somatotopic disorganization starts at around 4 months after suture, which seems to be somewhat slower than that in rats, and continues until a much later postoperative period. Furthermore, we suggested a possibility that the regeneration of one branch may affect the somatotopy of the unoperated nerve branch. These phenomena may contribute to aberrant facial nerve functions such as abnormal associated movement and facial spasm observed after nerve injury.     

Growth hormone treatment enhances the functional recovery of sciatic nerves after transection and repair

Introduction: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration. Methods: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance. Results: GH‐treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH. Conclusion: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair. Muscle Nerve, 2012     

自体肋间神经联合酸性成纤维细胞生长因子移植治疗高位脊髓损伤

背景：酸性成纤维细胞生长因子具有调节细胞增殖、移行、分化和生存的作用,也可以下调已知轴突再生的抑制因子如蛋白聚糖等,帮助轴突克服这些抑制因子,对神经纤维再生有重要作用。 目的：观察酸性成纤维细胞生长因子联合周围神经移植治疗大鼠高位脊髓损伤的可行性及效果。 方法：健康成年雌性SD大鼠108只随机抽签法分为自体神经组、自体神经联合生长因子组、高位脊髓横断组。咬除大鼠T8~10棘突、椎板,显露硬膜囊,水平切断高位脊髓并切除3 mm,显微镜下确认无神经纤维相连。自体神经组、自体神经联合生长因子组取双侧第8~10对肋间神经各2 cm,将肋间神经交叉移植入高位脊髓缺损处(近端白质与远端灰质、远端白质与近端灰质),分别以纤维蛋白凝胶、含有酸性成纤维细胞生长因子的纤维蛋白凝胶固定植入的肋间神经,缝合硬膜。高位脊髓横断组断端间旷置。术后90 d,行体感诱发电位及运动诱发电位检测观察神经电生理恢复情况。术后76 d,生物素葡聚糖胺顺行神经示踪观察运动传导束恢复情况。术后60 d,后肢BBB运动功能评分观察肢体运动恢复情况。 结果与结论：高位脊髓横断组大鼠均未引出体感及运动诱发电位波形。自体神经组、自体神经联合生长因子组均可引出体感及运动诱发电位,自体神经联合生长因子组体感诱发电位及运动诱发电位的平均潜伏期和波幅、BBB评分均明显优自体神经组(P < 0.01)。自体神经组和自体神经联合生长因子组在损伤区有较多生物素葡聚糖胺标记阳性神经纤维通过,明显多于高位脊髓横断组(P < 0.01),自体神经联合生长因子组多于自体神经组(P < 0.01)。提示自体周围神经移植酸性成纤维细胞生长因子能更好地恢复高位脊髓损伤后大鼠肢体运动功能。     

Effects of basic fibroblast growth factor (bFGF)-neutralizing antibody and platelet factor 4 on facial nerve regeneration

Exogenous basic fibroblast growth factor (bFGF) has been shown to prevent death of injured cholinergic neurons and stimulate neurite outgrowth from the proximal stump of the transected sciatic nerve. The present study was designed to examine the role of endogenous bFGF, rather than exogenous bFGF in the regenerative process of the transected facial nerve of guinea pig, by using the so-called silicone tubulization model which enabled us to bridge the transected facial nerve with a silicone tube and to inject into the tube bFGF-neutralizing antibody, normal IgG, saline, or platelet factor 4 (an antagonist for bFGF receptor). Under light microscopy, treatment with bFGF-neutralizing antibody caused significant decreases in vascular number, vascular area, and regenerating axons in the middle point of regeneration chambers at the third week after facial nerve transection, even though electron microscopy revealed that the bFGF-neutralizing antibody increased the number of thin axons with caliber smaller than 1 micrometer. Treatment with platelet factor 4 exhibited similar but more conspicuous effects on facial nerve regeneration. These findings suggest that endogenous bFGF not only facilitates angiogenesis within the transected facial nerve but also acts as a neurotrophic agent during facial nerve regeneration; it appears that endogenous bFGF contributes to the enlargement of axon caliber and increases the number of relatively large caliber axons.