脊髓损伤大鼠远端神经元及骨骼肌变化的基础研究

目的观察脊髓损伤大鼠远端神经元及骨骼肌变化情况。方法 20只大鼠随机分为2组,每组10只,分别为假手术组和脊髓损伤组,假手术组行椎板切除术,脊髓损伤组行胸10完全脊髓损伤,在制成模型后1、2、4、12、24周观察大鼠坐骨神经-运动终板-内侧腓肠肌形态变化情况。结果脊髓损伤组电镜下坐骨神经术后12周有髓神经纤维髓鞘崩解,其板层结构清晰,有髓神经纤维髓鞘于术后24周模糊、碎裂髓鞘变多,12周后无髓神经纤维及薄髓增多;术后12周腓肠肌光镜下局部肌细胞多数模糊,但边界清楚,结缔组织增生明显,肌细胞核相对聚集;肌细胞于术后24周融合,融合细胞间有空隙,细胞核密集,结缔组织增生明显;术后12周电镜下运动终板突触前后及皱褶膜不可分辨,肌纤维明暗带清晰,突触结构紊乱,z线不连续,高倍镜下突触前后膜不可辨,突触皱褶未见,可见类圆形细小颗粒及突触小泡,肌板结构清晰。结论大鼠脊髓损伤后在损伤平面以下周围神经、运动终板、骨骼肌在形态上会发生规律性变化,12周后显著变化,24周后则毁损性改变。     

骨骼肌卫星细胞异体移植对周围神经缺损后再生的影响

背景：有研究表明肌卫星细胞不仅在体外具有较强的增殖能力及适应能力,而且在异体内免疫原性低,免疫排斥反应低,移植后存活时间长。因此设想肌卫星细胞异体移植在促进缺损神经再生等方面可能具有良好的研究和应用前景。 目的：探讨骨骼肌卫星细胞移植对周围神经缺损后再生修复的影响。 方法：将16只Wistar大鼠随机分成移植组与对照组,每组8只,均切断右后肢坐骨神经,并通过生物可降解膜包裹缺损神经断端形成神经再生室。用微量注射器抽取已配制成的肌干细胞悬液0.2 mL注入移植组的神经再生室内。对照组注入等量的生理盐水。术后4,8和12周进行大鼠行步态测定,并用锇酸染色法制片观察缺损神经再生情况。观测大鼠坐骨神经功能指数、腓肠肌湿质量恢复率、再生的有髓神经纤维数量和直径及髓鞘厚度的变化。 结果与结论：大鼠经肌卫星细胞移植后腓肠肌湿质量残存率、再生的有髓神经纤维数目、直径及髓鞘厚度等项检测指标与对照组相比均差异有显著性意义(P < 0.05)。术后8和12周,移植组坐骨神经功能指数恢复情况明显优于对照组(P < 0.05)。实验结果提示在神经再生室中加入肌卫星细胞能促进缺损神经纤维的再生及其结构的成熟。 关键词：肌卫星细胞;生物可降解膜;异体;细胞移植;周围神经缺损;神经再生     

Combining acellular nerve allografis with brain- derived neurotrophic factor transfected bone marrow mesenchymal stem cells restores sciatic nerve injury better than either intervention alone

In this study, we chemically extracted acellular nerve allografts from bilateral sciatic nerves, and repaired 10-mm sciatic nerve defects in rats using these grafts and brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells. Experiments were performed in three groups： the acellular nerve allograft bridging group, acellular nerve allograft ＋ bone marrow mesenchymal stem cells group, and the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchyrnal stem cells group. Results showed that at 8 weeks after bridging, sciatic functional index, triceps wet weight recovery rate, myelin thickness, and number of myelinated nerve fibers were significantly changed in the three groups. Variations were the largest in the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells group compared with the other two groups. Experimental findings suggest that chemically extracted acellular nerve allograft combined nerve factor and mesenchymal stem cells can promote the restoration of sciatic nerve defects. The repair effect seen is better than the single application of acellular nerve allograft or acellular nerve allograft combined mesenchymal stem cell transplantation.     

Bridging peripheral nerves using a deacetyl chitin conduit combined with short-term electrical stimulation

Previous studies have demonstrated that deacetyl chitin conduit nerve bridging or electrical stimulation can effectively promote the regeneration of the injured peripheral nerve. We hypoth-esized that the combination of these two approaches could result in enhanced regeneration. Rats with right sciatic nerve injury were subjected to deacetyl chitin conduit bridging combined with electrical stimulation （0.1 ms, 3 V, 20 Hz, for 1 hour）. At 6 and 12 weeks after treatment, nerve conduction velocity, myelinated axon number, ifber diameter, axon diameter and the thickness of the myelin sheath in the stimulation group were better than in the non-stimulation group. The results indicate that deacetyl chitin conduit bridging combined with temporary electrical stimu-lation can promote peripheral nerve repair.     

Tacrolimus reduces scar formation and promotes sciatic nerve regeneration

A sciatic nerve transection and repair model was established in Sprague-Dawley rats by transecting the tendon of obturator internus muscle in the greater sciatic foramen and suturing with nylon sutures. The models were treated with tacrolimus gavage (4 mg/kg per day) for 0, 2, 4 and 6 weeks. Specimens were harvested at 6 weeks of intragastric administration. Masson staining revealed that the collagen fiber content and scar area in the nerve anastomosis of the sciatic nerve injury rats were significantly reduced after tacrolimus administration. Hematoxylin-eosin staining showed that tacrolimus significantly increased myelinated nerve fiber density, average axon diameter and myelin sheath thickness. Intragastric administration of tacrolimus also led to a significant increase in the recovery rate of gastrocnemius muscle wet weight and the sciatic functional index after sciatic nerve injury. The above indices were most significantly improved at 6 weeks after of tacrolimus gavage. The myelinated nerve fiber density in the nerve anastomosis and the sciatic nerve functions had a significant negative correlation with the scar area, as detected by Spearman’s rank correlation analysis. These findings indicate that tacrolimus can promote peripheral nerve regeneration and accelerate the recovery of neurological function through the reduction of scar formation.     

Platelet-rich plasma gel in combination with Schwann cells for repair of sciatic nerve injury

Bone marrow mesenchymal stem cells were isolated from New Zealand white rabbits, culture-expanded and differentiated into Schwann cell-like cells. Autologous platelet-rich plasma and Schwann cell-like cells were mixed in suspension at a density of 1 × 10 6 cells/mL, prior to introduction into a poly (lactic-co-glycolic acid) conduit. Fabricated tissue-engineered nerves were implanted into rabbits to bridge 10 mm sciatic nerve defects (platelet-rich plasma group). Controls were established using fibrin as the seeding matrix for Schwann cell-like cells at identical density to construct tissue-engineered nerves (fibrin group). Twelve weeks after implantation, toluidine blue staining and scanning electron microscopy were used to demonstrate an increase in the number of regenerating nerve fibers and thickness of the myelin sheath in the platelet-rich plasma group compared with the fibrin group. Fluoro-gold retrograde labeling revealed that the number of Fluo-ro-gold-positive neurons in the dorsal root ganglion and the spinal cord anterior horn was greater in the platelet-rich plasma group than in the fibrin group. Electrophysiological examination confirmed that compound muscle action potential and nerve conduction velocity were superior in the plate-let-rich plasma group compared with the fibrin group. These results indicate that autologous plate-let-rich plasma gel can effectively serve as a seeding matrix for Schwann cell-like cells to construct tissue-engineered nerves to promote peripheral nerve regeneration.     

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.     

Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice

We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.     

失神经骨骼肌细胞凋亡：运动与感觉神经的比较

Skeletal muscle atrophy inevitably occurs in denervated skeletal muscle,and cell apoptosis plays an important role in skeletal muscle atrophy and degeneration.The present study established rat models of simple nerve injury by transecting the ventral or dorsal spinal nerve root and observed rat skeletal muscle cell apoptosis following simple motor nerve injury versus simple sensory nerve injury.Following skeletal muscle denervation for 10 weeks,cell apoptosis was detected in skeletal muscle,which was accompanied by obvious changes in rat behavior and electrophysiological responses.In addition,changes in cross-sectional area and average gray-scale of motor endplates of the gastrocnemius muscle were analyzed following sciatic nerve injury and motor nerve injury.Cell nuclei in denervated skeletal muscle tissue were more densely arranged than in normal skeletal muscle tissue.Cell nuclei were most dense in the sciatic nerve injury group,followed by the motor nerve injury group and the sensory nerve injury group.Fas/FasL expression and the number of apoptotic cells increased in denervated skeletal muscle,and apoptosis-related changes were observed.These findings suggested that motor and sensory nerves provided trophic actions following skeletal muscle and motor nerve injury,resulting in a greater influence on skeletal muscle atrophy than sensory nerve injury.Therefore,reconstruction of motor nerves should be preferentially considered for treating denervation-induced skeletal muscle atrophy.     

Regeneration of mouse peripheral nerves in degenerating skeletal muscle: guidance by residual muscle fibre basement membrane

The part played by basement membrane in the guidance of peripheral nerve growth in vivo has been assessed by examining the capacity of degenerating mouse muscle to support the regeneration of the cut sciatic and saphenous nerves. Ethanol and formaldehydefixed gluteus maximus muscles were implanted around the contralateral cut nerves. The subsequent nerve growth into the degenerating muscle was assessed by silver staining after 3, 4 and 10 days. By 4 days, linear axonal growth was seen, parallel to the length of the muscle fibers, and coinciding with the onset of degeneration of the sarcoplasm. Transverse sections of the 10 day preparations showed that over 90% of linearly growing axons were located inside the remaining sheaths of muscle fibre basement membrane. This relationship was confirmed by electron microscopy of ruthenium red-stained preparations. Both motor and sensory axons were able to grow in this manner, for electrophysiological testing revealed the presence of motor axons from the sciatic nerve, while the saphenous nerve contains only sensory axons. Identical growth was seen at 10 days in muscles caused to degenerate by incubation in distilled water. However, linear growth did not occur in live-innervated and glutaraldehyde-fixed muscles, in which muscle fibre architecture was preserved.It is concluded that basement membrane derived from muscle can promote peripheral nerve regeneration. Furthermore, both motor and sensory axons show a strong preference for growth along its inner surface, the basal lamina.     

Nanofibrous nerve conduits for repair of 30-mm-long sciatic nerve defects*

It has been confirmed that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit can promote peripheral nerve regeneration in rats. However, its efficiency in repair of over 30-mm-long sciatic nerve defects needs to be assessed. In this study, we used a nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit to bridge a 30-mm-long gap in the rat sciatic nerve. At 4 months after nerve conduit implantation, regenerated nerves were macroscopically observed and histologically assessed. In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. Masson’s trichrome staining showed that there were no pathological changes in the size and structure of gastrocnemius muscle cells on the operated side of rats. These findings suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects.     

Cholesterol reutilization during myelination of regenerating PNS axons is impaired in Niemann-Pick disease type C mice

Niemann-Pick C (NPC) disease is an autosomal recessive lipidosis characterized by lysosomal accumulations of unesterified cholesterol. Cultured NPC cells exhibit a defect in the intracellular trafficking of LDL-derived cholesterol that leads to lysosomal accumulations of unesterified cholesterol. We found in a preliminary study that the myelination of regenerating axons was retarded in the NPC mouse following sciatic nerve crush. Because lipoprotein-mediated cholesterol transport is involved in myelination during nerve regeneration, we investigated whether this cholesterol reutilization pathway was perturbed in the NPC mouse. Mice received intraneural injections of [³H]acetate to label myelin cholesterol, and 2 weeks later the injected nerves were crushed above the injection site. Four weeks after crush, the nerves were examined by electron microscopic autoradiography. In normal mice, regeneration was well advanced, with thick myelin sheaths surrounding the regenerated axons and very little myelin debris remaining. The new myelin sheaths were well labeled, indicative of efficient cholesterol reutilization. In NPC mice, the new myelin sheaths were thinner and contained little label, indicative of retarded regeneration and little or no cholesterol reutilization. These data suggest the possibility of a causal link between compromised cholesterol reutilization and delayed or slowed regeneration of myelin sheaths. J. Neurosci. Res. 49:389–392, 1997. © 1997 Wiley-Liss, Inc.     

Ganglioside promotes the bridging of sciatic nerve defects in cryopreserved peripheral nerve allografts

Previous studies have shown that exogenous gangliosides promote nervous system regeneration and synapse formation.In this study,10 mm sciatic nerve segments from New Zealand rabbits were thawed from cryopreservation and were used for the repair of left sciatic nerve defects through allograft bridging.Three days later,1 m L ganglioside solution(1 g/L) was subcutaneously injected into the right hind leg of rabbits.Compared with non-injected rats,muscle wet weight ratio was increased at 2–12 weeks after modeling.The quantity of myelinated fibers in regenerated sciatic nerve,myelin thickness and fiber diameter were elevated at 4–12 weeks after modeling.Sciatic nerve potential amplitude and conduction velocity were raised at 8 and 12 weeks,while conduction latencies were decreased at 12 weeks.Experimental findings indicate that ganglioside can promote the regeneration of sciatic nerve defects after repair with cryopreserved peripheral nerve allografts.     

Effects of tacrolimus (FK506) on sciatic nerve regeneration in rats

BACKGROUND: Tacrolimus (FK506) protects peripheral nerves located in damaged regions by inhibiting T lymphocyte proliferation and activation.OBJECTIVE: To evaluate the effect of FK506 on promoting regeneration of rat sciatic nerve. DESIGN, TIME AND SETTING: A randomized, controlled, animal study was performed at the Laboratory of the Department of Orthopedic Surgery, Dalian Medical University, China, from September 2007 to September 2008.MATERIALS: A total of 60 adult, male, Sprague-Dawley rats were equally and randomly divided into model, local administration and systemic administration groups. All rats received a neurotomy of bilateral sciatic nerves to establish models of nerve regeneration chambers. The powder and injection of FK506 were supplied by Fujisawa Pharmaceutical, Japan.METHODS: The regeneration chambers of the model group were infused with 0.2 mL saline. The systemic group were injected with 0.2 mL saline, followed by daily subcutaneous injections of FK506 (1 mg/kg), for 14 days. The local administration group was infused with 0.2 mL FK506 (1 μg/mL).MAIN OUTCOME MEASURES: Local immune response was observed using hematoxylin-eosin staining. Myelinated nerve fiber number, myelin sheath and nerve fiber thickness were observed using toluidine blue staining. Wet weight of gastrocnemius was evaluated. Compound muscle action potential amplitude, latency, and conduction time were recorded, and motor nerve conduction velocity was calculated using electrophysiology.RESULTS: The total number of myeiinated nerve fibers in the local and systemic administration groups was significantly higher than in the model group. The density of myelinated nerve fibers, myelin sheath thickness and mean axon diameter were significantly increased in the systemic administration group compared with the model group (P < 0.05). Lymphocyte infiltration was decreased in the local and systemic administration groups compared with the model group. The wet weight of rat gastrocnemius in the local and systemic administration groups were significantly greater compared with the model group (P<0.05). Motor nerve conduction velocity was the fastest in the systemic administration group, and the slowest in the model group. Compound muscle action potential amplitude was larger in the systemic administration group compared with the local administration and model groups (P<0.05).CONCLUSION: Systemic administration of FK506 can promote regeneration of rat sciatic nerve and recovery of neural function. Systemic administration produced better regeneration and recovery of function than local administration of FK506.     

骨髓间充质细胞构建组织工程神经修复坐骨神经缺损

背景：许旺细胞是周围神经组织工程的种子细胞,但体外分离、培养、纯化许旺细胞较困难。脱细胞同种异体神经移植物具有较强的修复外周神经缺损的能力,且可诱导骨髓间充质细胞分化为类许旺细胞,理论上骨髓间充质细胞可替代许旺细胞作为种子细胞应用于周围神经组织工程。 目的：观察骨髓间充质细胞构建组织工程神经修复坐骨神经缺损的效果,评估骨髓间充质细胞作为种子细胞修复周围神经缺损的可行性。 设计、时间及地点：随机对照动物实验,于2008-07/12在大理学院基础医学院实验室完成。 材料：将30只SD大鼠按随机数字表法分为3组,每组10只。骨髓间充质细胞+异体移植组将骨髓间充质细胞复合脱细胞同种异体神经移植物培养的组织工程神经与两断端用10/0 无创线端端吻合;异体移植组将脱细胞同种异体神经移植物桥接;自体移植组将切断的坐骨神经旋转180°端端吻合。 方法：运用骨髓间充质细胞构建的组织工程神经修复大鼠10 mm坐骨神经缺损,移植后12周通过坐骨神经功能指数、腓肠肌湿质量恢复率、S-100免疫组织化学染色、电镜等方法观察移植物修复效果。 主要观察指标：复合物培养时观察细胞形态的变化;移植后观察坐骨神经功能指数及腓肠肌湿质量恢复率;通过甲苯胺蓝染色观察新生髓鞘形成和轴突生长及神经纤维的分布情况,结合透射电镜及S-100蛋白免疫组织化学染色,观察许旺细胞生长和神经纤维再生情况。 结果：坐骨神经功能指数及腓肠肌湿质量恢复率的检测结果显示骨髓间充质细胞+异体移植组优于异体移植组(P < 0.05)。骨髓间充质细胞+异体移植组复合物中S-100的表达明显高于异体移植组,有髓神经纤维数量、有髓纤维直径和髓鞘厚度均大于异体移植组(P < 0.05),修复效果接近自体移植组。 结论：骨髓间充质细胞构建的组织工程神经修复周围神经缺损的效果优于单纯的脱细胞同种异体神经移植物,骨髓间充质细胞作为种子细胞在周围神经组织工程中具有较强的应用价值。     

Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats

Human umbilical cord mesenchymal stem cells,incorporated into an amnion carrier tubes,were assessed for nerve regeneration potential in a rat nerve defect model.Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell (cell transplantation group)or saline(control group).At 8,12,16 and 20 weeks after cell implantation,the sciatic functional index was higher in the cell transplantation group compared with the control group.Furthermore,electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased.Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time.Gastrocnemius muscle cell diameter,wet weight and restoration ratio were increased.These data indicate that transplanted human umbilical cord mesenchymal stem cells,using the amnion tube connection method,promote restoration of damaged sciatic nerves in rats.     

Repair of sciatic nerve defects using tissue engineered nerves*

In this study, we constructed tissue-engineered nerves with acellular nerve allografts in Sprague-Dawley rats, which were prepared using chemical detergents-enzymatic digestion and mechanical methods, in combination with bone marrow mesenchymal stem cells of Wistar rats cultured in vitro, to repair 15 mm sciatic bone defects in Wistar rats. At postoperative 12 weeks, electrophysiological detection results showed that the conduction velocity of regenerated nerve after repair with tissue-engineered nerves was similar to that after autologous nerve grafting, and was higher than that after repair with acellular nerve allografts. Immunohistochemical staining revealed that motor endplates with acetylcholinesterase-positive nerve fibers were orderly arranged in the middle and superior parts of the gastrocnemius muscle; regenerated nerve tracts and sprouted branches were connected with motor endplates, as shown by acetylcholinesterase histochemistry combined with silver staining. The wet weight ratio of the tibialis anterior muscle at the affected contralateral hind limb was similar to the sciatic nerve after repair with autologous nerve grafts, and higher than that after repair with acellular nerve allografts. The hind limb motor function at the affected side was significantly improved, indicating that acellular nerve allografts combined with bone marrow mesenchymal stem cell bridging could promote functional recovery of rats with sciatic nerve defects.     

Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury

Medialization thyroplasty or injection laryngoplasty for unilateral vocal fold paralysis cannot restore mobility of the vocal fold. Recent studies have shown that transplantation of mesenchymal stem cells is effective in the repair of nerve injuries. This study investigated whether adipose-derived stem cell transplantation could repair recurrent laryngeal nerve injury. Rat models of recurrent laryngeal nerve injury were established by crushing with micro forceps. Adipose-derived mesenchymal stem cells(ADSCs; 8 × 105) or differentiated Schwann-like adipose-derived mesenchymal stem cells(d ADSCs; 8 × 105) or extracellular matrix were injected at the site of injury. At 2, 4 and 6 weeks post-surgery, a higher density of myelinated nerve fiber, thicker myelin sheath, improved vocal fold movement, better recovery of nerve conduction capacity and reduced thyroarytenoid muscle atrophy were found in ADSCs and d ADSCs groups compared with the extracellular matrix group. The effects were more pronounced in the ADSCs group than in the d ADSCs group. These experimental results indicated that ADSCs transplantation could be an early interventional strategy to promote regeneration after recurrent laryngeal nerve injury.     

Effects of sciatic nerve regeneration on axonal populations in tributary nerves

The present study tests 2 hypotheses: (1) that the numbers of axons that regenerate into a tributary nerve are in part dependent on the type of lesion used to transect the axons in the parent nerve; and (2) that the numbers of axons that regenerate will be different in different tributary nerves. Axons were counted in the sural nerve and the nerve to the medial gastrocnemius muscle 8 weeks following crush, simple transection, transection with removal of 4 mm and transection with removal of 8 mm of the sciatic nerve in the rat. The counts of myelinated and unmyelinated axons are presented in the text. If axon numbers in the 2 nerves are normalized, the proportion of regenerated to normal myelinated axon numbers are approximately the same in the 2 nerves, with more regenerated axons than normal following crush, simple transection, or 4 mm gap transection and fewer following 8 mm gap transection. The unmyelinated axons behave differently. In the nerve to the medial gastrocnemius muscle, the numbers of unmyelinated axons are greater than or equal to the normal numbers following our various surgical paradigms whereas in the sural nerve there are always fewer unmyelinated axons than normal. These findings indicate that the above hypotheses are correct for the nerves tested in the rat.     

Development of myelinated nerve fibers in the sixth cranial nerve of the rat: a quantitative electron microscope study

Myelination was studied quantitatively in the sixth cranial nerves of rats by counting and measuring all myelinated fibers during the first three postnatal weeks. In transverse semithin and thin sections cut serially at a well-defined anatomical site in the midsphenoid region, only a few axons (mean 12) were myelinated at birth. On days 2, 4, and 8, counts of myelinated fibers were respectively 5 times (mean 57), 20 times (mean 230), and 24 times (mean 273) the number seen at birth. During the second postnatal week, the number of myelinated fibers remained constant, whereas growth of axons and their myelin sheaths continued. By 15 days these fibers were large and relatively uniform in size; they had compact, circular myelin sheaths. During the third postnatal week, myelination of previously unmyelinated, smaller axons began. The number of myelinated fibers increased again and the size distribution of myelinated fibers became bimodal. Axon diameters, fiber diameters, and myelin sheath dimensions for all fibers were calculated from measurements made on electron micrographs. The transverse length of the myelin membrane increased exponentially with time. The growth increased rapidly during the formation of the first 20 spiral layers and remained relatively constant during the subsequent enlargement of the compact sheath. The association of axon diameter and myelin sheath thickness was poor at young ages, but it improved progressively with maturation of the sheath. The results show that myelination begins around axons that have a wide range of diameters. Also, the first axons to be myelinated become the large myelinated fibers of the sixth nerve. The small myelinated fibers originate from axons that do not become myelinated until the third postnatal week. Myelination, though differing in onset by 2 weeks, appeared to be similar in both populations as judged by similarity of sheath morphology and growth rates. It is of interest that at the level studied, the sixth nerve also contains a fascicle of unmyelinated cranial sympathetic fibers.