Acceleration of peripheral nerve regeneration after crush injury in rat

It the sciatic nerve of a rat is crushed in the thigh, axons from the proximal side of the crush will regenerate so that the toe-spreading reflex becomes observable again after 10.4 +/- 1.7 (mean +/- S.D.) days. If the nerve is electrically stimulated for 0.25-1.0 h at the crush site, just after the crush occurs, the toe-spreading reflex first becomes observable 4.14 +/- 1.6 (mean +/- S.D.) days after the crush. Stimulation is most effective if delivered immediately after the crush but can be delayed up to an hour and still cause significantly faster regeneration. This phenomenon could be useful in clinical management of crushed peripheral nerves.     

Peripheral and central nervous system lesions caused by triethyl- and trimethyltin salts in rats

Both trimethyltin and triethyltin salts are known to produce toxic lesions in the central nervous system. Triethyltin intoxication has been associated with central intramyelin edema, while trimethyltin has been shown to produce neuronal necrosis in selected limbic and sensory regions of the brain. Only scant attention has been paid to peripheral nerves of animals treated with alkyltins. In this study, we have treated rats with 6 or 8 mg/kg trimethyltin, and 1, 2, 4, 6, or 8 mg/kg triethyltin (single or multiple exposure), and evaluated in detail at the light microscope level both central and peripheral nervous system lesions. In addition to the central neuron necrosis or myelin edema described previously, both compounds produced peripheral axon degeneration and chromatolysis of large spinal cord and brain stem neurons. Chromatolysis was seen in reticular neurons of the brain stem and ventral horn or spinal cord in rats receiving high doses (6 or 8 mg/kg) of triethyltin, and in these same areas plus mesencephalic trigeminal nucleus in animals treated with trimethyltin. Wallerian-like degeneration of peripheral axons was seen in sciatic and tibial nerve and ventral roots of animals receiving 3 injections of 4 mg/kg or single or multiple injections of 6 or 8 mg/kg triethyltin. Axon degeneration was also seen in sciatic and tibial nerves 21 days after a single exposure to 8 mg/kg trimethyltin. Since myelin edema is believed to be reversible, the axonal changes described here may be of greater clinical significance in relation to human exposure.     

He—Ne激光对受损感觉神经纤维轴浆运输作用的实验研究

大鼠坐骨神经干被夹伤后,立即用剂量为51J/cm~2的He-Ne激光照射损伤部位。作者发现由损伤部位远侧引入的辣根过氧化物酶经轴浆运输标记同侧腰4—6脊神经节中的大型神经元胞体数较对照明显增加(P<0.001)。结果提示大鼠坐骨神经夹伤早期,激光照射有利于维持神经中较粗感觉纤维的轴浆运输。     

pro-BDNF在初级感觉神经元内的顺行和逆行输送

摘　要　研究表明脑源性神经营养因子(BDNF)在神经元内可顺行运输至末梢并释放到下一级神经元参与突触可塑性等功能。而成熟的BDNF由其前体分子(pro -BDNF)经蛋白酶水解形成。体外研究表明pro -BDNF可以激活神经营养因子受体TrkB并导致TrkB的磷酸化,但pro -BDNF在体内的作用目前仍不清楚。本文拟探讨内源性pro -BDNF在周围神经的运输。将大鼠坐骨神经结扎或压榨背根制成模型,动物存活不同时间后取坐骨神经、背根神经节和脊髓进行pro -BDNF免疫组织化学染色检测。结果显示:pro- BDNF免疫阳性产物沉积于坐骨神经和背根损伤处的近侧端和远侧端, 24h达高峰,近侧端可持续达 7d而远侧端可达3d;在坐骨神经的近侧端和远侧端、背根神经节和脊髓可检测到全分子量大小的完整pro -BDNF;在转染pro- BDNF质粒后,PC12细胞内可见pro -BDNF免疫阳性产物分布于胞体和突起。这些结果提示pro- BDNF可以象成熟BDNF一样在感觉神经内顺行和逆行运输,并可由神经元分泌、释放而发挥生理作用。     

A scanning electron microscopic study of peripheral nerve degeneration and regeneration

The left sciatic nerves of male Holtzman rats were exposed and crushed with a hemostat for 3 s at a point midway between the inferior gluteal nerve and the bifurcation into the tibial and common peroneal nerves. Normal myelinated axons appear in the scanning electron microscope as relatively smooth cylindrical structures with interweaving strands of collagen fibers coursing over their length. Nodes of Ranvier are seen as constrictions along the myelinated fibers. After crushing, the nerve fibers swell, and interruptions appear in the nodal and internodal regions causing a beaded morphology. The myelin sheaths fragment into numerous spherical bodies as the degenerative process progresses. Some remnants of myelin debris are found within the nerves 60 days following the lesion A few regenerating myelinated axons are observed within the connective tissue fibers and myelin debris distal to the crushed regions approx 15 days after placement of the lesion. By day 30 the nerves are relatively normal in appearance. The scanning electron micrographs provide a three-dimensional picture of the dynamic processes which occur during nerve degeneration and regeneration. These findings are correlated with previous studies and can serve as a basis for future scanning electron microscopic studies in this area of research.     

Localization and axonal transport of immunoreactive cholinergic organelles in rat motor neurons—An immunofluorescent study

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated α-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors.The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: (1) Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. (2) Deafferentation did not notably influence accumulations of immunoreactive material. (3) Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. (4) Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots.It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

Electrical stimulation of nerve regeneration in the rat: the early effects evaluated by a vibrating probe and electron microscopy

This study examines the effect of applied d.c. electric fields on nerve regeneration following injury to the rat sciatic nerve using the circularly vibrating probe and electron microscopy. The transected and treated nerve which received a d.c. electrical stimulator (0.6 mu A) was compared with untreated transected and crushed nerves. At one week postoperative, the probe was used to measure in vivo the current density along the nerve length. All nerves studied had a proximal peak at the lesion site and a second peak at varying distal locations: crushed/untreated (13.3 mm), transected/untreated (9.7 mm) and transected/treated (16.3 mm). A significant difference (69%) between the distal peak distances in the two transection groups suggests that the electrical treatment enhanced the progress of nerve regeneration. There were no significant differences between the mean peak amplitudes (1.6-2.2 mu A/cm2). Applied verapamil reduced the peaks, suggesting they are associated in part with a calcium-dependent current. Electron microscopy at selected nerve regions indicated that the peaks correspond to regenerating axonal growth cones. The results suggest the potential clinical application of d.c. electric fields in the treatment of nerve injuries.     

靶肌肉注射促红细胞生成素对大鼠周围神经再生的作用

目的探讨靶肌肉注射人重组促红细胞生成素（recombinant human erythropoietin，rh-EP0）对大鼠坐骨神经损伤后神经再生的作用。方法选用健康雄性SD大鼠12只，制备大鼠右侧坐骨神经钳夹损伤模型。实验动物随机分为2组，每组6只，EPO组：靶肌肉注射rh-EPO2500U／kg；对照组：注射同体积的生理盐水。术后第7d、14d、21d观察坐骨神经功能指数（SFI），第21d组织学观察脊髓腰膨大（L4～L6）、夹伤远端坐骨神经、损伤侧腓肠肌组织并作图象分析测定脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标。结果术后第7d两组SFI无显著性差异，术后第14d、21dEPO组SFI恢复程度明显大于对照组，差别有显著性意义（P〈0．05）；术后第21d损伤侧脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标，EPO组均优于对照组（P〈0．01，P〈0．05）。结论靶肌肉注射rh-EPO能促进周围神经再生和功能恢复。     

Effects of gangliosides on the methionine uptake in crushed sciatic nerves of rats with alloxan diabetes

Effect of gangliosides on the transmembrane transport of methionine in crushed sciatic nerves was studied in alloxan diabetic and control rats until the 4th day after nerve crushing. Methionine uptake rate decreases while time passes after injury in both control and diabetic rats, but the decrement is greater in diabetic rats than in controls. Ganglioside treatment causes an enhancement on the transmembrane transport in both diabetic and control animals, but the effect in time was greater in diabetic rats than in controls.     

Organization of peripheral nerves and spinal roots of the Atlantic stingray, Dasyatis sabina

1. The sizes and numbers of axons in peripheral nerves and spinal roots were investigated in the stingray, Dasyatis sabina. 2. The axons of the dorsal and ventral roots do not mingle in peripheral nerves of this animal as they do in higher vertebrates. Thus, it was usually possible to split the peripheral nerve into two portions, one containing only dorsal root axons, the other containing only ventral root axons. This feature was useful for the analysis of certain aspects of spinal cord organization. 3. The fact that dorsal and ventral root axons were segregated in peripheral nerves enabled us to demonstrate, without experimental surgery, that the central processes of the dorsal root ganglion cells and the proximal ventral root axons were 10-20% narrower, on the average, than the distal processes of the same dorsal root ganglion cells or the distal parts of the same ventral root axons. 4. The stingray is remarkable in having very few unmyelinated axons in the dorsal roots, ventral roots, or peripheral nerves. This paucity of unmyelinated axons distinguishes the Atlantic stingrays from all other vertebrates whose roots and nerves have been examined for unmyelinated fibers. 5. Similar findings were obtained for one spotted eagle ray (Aetobatus narinari) and two cow-nose rays (Rhinoptera bonasus).     

The distribution of somatomedins in the nervous system of the cat and their release following neural stimulation

Recent evidence suggests a regulatory role in the nervous system for somatomedins. The present study, using a somatomedin radioreceptorassay which primarily detects insulin-like growth factors 1 and 2, shows that somatomedins are widely distributed throughout the nervous system of the cat. Whilst somatomedins were present in all CNS regions, the highest concentration occurred in the hypothalamus followed by cerebral cortex. In the spinal cord, the dorsal roots contained twice the concentration found in the ventral roots. Activity was also present in the sympathetic ganglia, vagus nerve and sciatic nerves. Following electrical stimulation of the brachial and sciatic nerves somatomedins were released into perfusates from extirpated cut limbs. These findings suggest that somatomedins may be neuroregulatory hormones.     

β-1,4半乳糖基转移酶-I在钳夹伤后大鼠坐骨神经中表达的变化

为了观察β-1,4-半乳糖基转移酶-I(β-1,4-galactosyltransferase-I,β-1,4-GalT-I)在正常和钳夹伤后大鼠坐骨神经中表达的变化,本研究采用RT-PCR方法,从小鼠坐骨神经中特异性地扩增β-1,4-GalT-I cDNA片段并将其克隆到pGEM-T载体。采用体外转录的方法合成地高辛标记的正、反义β-1,4-GalT-I RNA探针。通过原位杂交和图像分析的方法,观察β-1,4-GalT-I mRNA在正常和夹伤大鼠坐骨神经中的表达及其变化。结果表明:β-1,4-GalT-I mRNA在大鼠坐骨神经的髓鞘中表达,在夹伤坐骨神经后1~2d,β-1,4-GalT-I mRNA在坐骨神经的表达最高,于夹伤后1周开始下降,在夹伤后1个月恢复至正常水平。上述结果提示坐骨神经夹伤后β-1,4-GalT-I mRNA的表达发生变化,并且主要表达在坐骨神经的Schwann细胞。本研究结果为进一步分析β-1,4-GalT-I在周围神经再生中的调控机制奠定了基础。     

GDNF及dvHSV-GDNF对坐骨神经损伤大鼠脊髓前角运动神经元的影响

为了探讨胶质细胞源性神经营养因子及单纯疱疹病毒载体介导的胶质细胞源性神经营养因子 (dv HSV-GDNF)对坐骨神经损伤大鼠脊髓前角运动神经元的作用 ,本实验对成年大鼠造成双侧坐骨神经损伤后 ,于右侧损伤处分别施加胶质细胞源性神经营养因子和 dv HSV-GDNF;左侧损伤处施加生理盐水作为对照。分别取损伤后 4、7、14和 2 8d大鼠的脊髓 L4 ～ L6 节段 ,经石蜡包埋切片后行 Nissl染色 ,计数前角运动神经元数量并进行统计学分析。结果发现 :坐骨神经损伤后 4、7、14和 2 8d,右侧脊髓前角运动神经元的数量明显高于左侧。提示 :胶质细胞源性神经营养因子和 dv HSV-GDNF可减少坐骨神经损伤大鼠脊髓前角运动神经元的死亡     

The effect of 2,5-hexanedione on the induction of ornithine decarboxylase in the dorsal root ganglion of the rat

Rat dorsal root ganglia respond to sciatic nerve injury with an increase in the activity of the enzyme ornithine decarboxylase (ODC). The increase is impaired under certain conditions (e.g. diabetes, Vinca alkaloid treatment) where retrograde axonal transport is reduced. The purpose of the experiments was to determine if the neurotoxin 2,5-hexanedione, also known to interfere with retrograde axonal transport, similarly affected ODC induction. Rats were treated with 2,5-hexanedione i.p. to a cumulative dose of 6 and 8 g/kg. One sciatic nerve was crushed under anaesthesia and 24 h later the dorsal root ganglia were removed and assayed for ODC activity by a radioenzymatic method. The ratio of ODC activity of 1.57 +/- 0.58 (crushed side over control side) was reduced to 1.02 +/- 0.41 1.08 +/- 0.39 after 2,5-hexanedione at 6 g and 8 g/kg, respectively. The enzyme was not inhibited by addition of 2,5-hexanedione in vitro. The results confirm the role of retrograde axonal transport in nerve cell responses to injury and are consistent with the effects of 2,5-hexanedione on nerve regeneration.     

The release of acetylcholine from the spinal cord of the cat by antidromic stimulation of motor nerves

1. ACh was measured in the effluent from the perfused lumbosacral cord of the cat with or without stimulation of the central ends of the cut left sciatic and femoral nerves after section of the left dorsal roots.

2. In about 30% of the preparations ACh was obtained in the samples collected at rest (average 3·3 ng/min); the amount of ACh release was increased 1·3-9 times by stimulation of the peripheral nerves. The average amount of ACh collected during stimulation of the peripheral nerves at 5/sec was 6·9 ng/min. Antidromic motor nerve impulses responsible for the ACh release were likely to be only those in alpha motor fibres.

3. There was a depression in ACh release/stimulus as the stimulus frequency was increased more than 10/sec. Such changes in ACh release with various stimulus frequencies were correlated with depression in the response of Renshaw cells to excitation through motor-axon collaterals.

4. Amounts of ACh release during stimulation of the peripheral nerves at 5/sec were significantly increased for 1 or 2 min after a short tetanic stimulation of the nerves.

5. Intravenous injection of dihydro-β-erythroidine did not reduce the amount of ACh release produced by stimulation of the peripheral nerves.

6. It is concluded that antidromic impulses in alpha motor fibres liberate ACh from the presynaptic terminals at the central synapses on Renshaw cells.

     

Induction of extrajunctional acetylcholine sensitivity of rat EDL muscle by peptidic component of peripheral nerve

The induction of extrajunctional ACh-sensitivity was studied electrophysiologically 4 days after implantation of segments of the sciatic nerve, segments of dorsal and ventral spinal roots and silastic plates, containing some components of peripheral nerve onto surface fibres of innervated rat extensor digitorum longus muscle. It has been found that extrajunctional ACh-sensitivity can be induced by a piece of rat sciatic nerve and by peptidic material of about 8,000 and 10,000 mol. wt.No sensitivity was induced by segments of ventral and dorsal roots, by a piece of rabbit sciatic nerve and by rat sciatic nerve sectioned 7 days prior to implantation.The active nerve fractions lost its inducing potency after pepsin pre-treatment and irradiation with UV-light. Peripheral nerve probably contains some specific factor, peptidic in nature, which is able to induce the ACh-sensitivity of muscle fibre membrane.     

脊髓小胶质细胞反应性在大鼠周围神经再生中的作用(英文)

为探讨大鼠坐骨神经损伤后脊髓小胶质细胞反应性、脊髓腹角运动神经元脱失与坐骨神经再生之间的关系,制备了SD大鼠右侧坐骨神经钳夹损伤模型,术后3d和7d测定相应脊髓节段小胶质细胞免疫反应性、腹角运动神经元数量,4周时于光镜和电镜下评价坐骨神经变性和再生。结果显示:(1)坐骨神经损伤后3d,脊髓腹角小胶质细胞OX-42免疫反应性开始明显增强(P<0.05);(2)脊髓腹角损伤同侧与对侧运动神经元数量比明显降低(P<0.05),说明同侧运动神经元存活数量减少;(3)组织学评价显示损伤神经再生不良;(4)simvastatin(一种降胆固醇药物,具有潜在的免疫调节作用)干预组较非simvastatin干预组小胶质细胞进一步激活,运动神经元存活数量增加,坐骨神经再生良好。本研究结果提示,脊髓腹角小胶质细胞的激活可能在大鼠周围神经损伤后的再生中发挥重要的保护作用。     

Rapid transport of noradrenaline in adrenergic axons of rat sciatic nerve distal to a crush.

The transport of NA in the rat sciatic nerve distal to a crush was studied in 5 mm segments. The disappearance of a NA fraction in consecutive segments with time after crushing (0-12 h) was interpreted to indicate a proximo-distal migration of a NA fraction into further distal parts of the nerve. This transportable NA fraction was found to be about 45% of the NA in normal sciatic nerves and migrated at a rate of approximately 8 mm/h. The apparently non-mobile fraction (55%) was probably mainly located in vaso-constrictor nerve terminals of the blood vessels supplying the nerve. This non-mobile NA fraction does not contribute to the NA accumulations proximal to a crush. Thus, when calculating the rate of transport of any substance from accumulation experiments, corrections for a non-mobile fraction must always be considered.     

An outflow of acetylcholine from normal and regenerating ventral roots of the cat

1. An outflow of acetylcholine (ACh) has been measured from cat ventral roots in situ and in vitro, using standard bio-assay techniques. The ACh was collected in chambers, filled with physiological solution containing anticholinesterase, which surrounded the root.

2. The outflow fell rapidly at first and then progressively more slowly, taking several hours to fall to a level near the assay threshold. The outflow of ACh resulted from cutting a root or from such conventional manipulations as stretching the root or teasing apart root filaments. The outflow could occur either from the end of a root (following cutting) or from along the length of the root (following stretching or teasing). The outflow was only detected in the presence of an anticholinesterase.

3. ACh was resynthesized by roots at a rate which was usually rapid enough to maintain their ACh content at its original level in spite of a considerable ACh outflow. In roots subjected to a minimum of interference their ACh content built up to about twice the initial content after 5-7 hr soaking in eserinized Krebs solution although the total ACh outflow was equivalent to the original ACh content.

Roots which were cut or stretched several times within a few hours put out a large amount of ACh and showed a fall in their ACh content below the initial level.

4. The time course of sealing of the damaged ends of axons following crushing was assessed by measuring the injury potential for 1-2 hr after crushing at room temperature; in normal roots the half-time for decline of the injury potential was 91 ± 10 min. This was slower than the rate of decline of the ACh outflow.

5. An explanation for the observed outflow of ACh is discussed in terms of diffusion of ACh and sealing of axon ends. It is concluded that much of the ACh in normal ventral root axons is present in a readily diffusible form.

6. Similar experiments on roots regenerating 9-10 days after crushing showed that the ACh outflow from regenerating roots was much smaller than from normal axons, although their ACh content was similar. This could have been due to more rapid sealing of the cut regenerating axons since the half-time for the decline of the injury potential following crushing of the regenerating axons was less than half that in control roots. The alternative possibility is considered that a high proportion of the ACh might be much less diffusible than in normal axons.

7. The subcellular distribution of ACh in normal and regenerating peripheral axons is discussed.