Chemical and structural changes of neurofilaments in transected rat sciatic nerve

The sequence of changes occurring in transected rat sciatic nerve was examined by electron microscopy and by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. Representative segments of transected nerves were processed for ultrastructural examinations between 0 and 34 days after the transection of sciatic nerves immediately below the sacro-sciatic notch. The remainder of the transected nerves and the intact portions of sciatic nerves were desheathed and immediately homogenized in 1 percent SDS containing 8 M urea and 50 mM dithioerythritol. Solubilized proteins were analyzed on 12 percent gels at pH 8.3 in a discontinuous electrophoretic system. Initial changes were limited to the axons of transected nerve fibers and were characterized by the loss of microtubules and neurofilaments and their replacement by an amorphous floccular material. These changes became widespread between 24 and 48 h after transection. The disruption of neurofilaments during this interval occurred in parallel with a selective loss of 69,000, 150,000 and 200,000 mol wt proteins from nerve homogenates, thus corroborating the view that these proteins represent component subunits of mammalian neurofilaments. Furthermore, the selective changes of neurofilament proteins in transected nerves indicate their inherent lability and suggest their susceptibility to calcium-mediated alterations. Electrophoretic profiles of nerve proteins during the 4-34-day interval after nerve transection reflected the breakdown and removal of myelin, the proliferation of Schwann cells and the deposition of endoneurial collagen. A marked increase of intermediate-sized filaments within proliferating Schwann cell processes was not accompanied by the appearance of neurofilamentlike proteins in gels of nerve homogenates.     

Microtubules in myelinated and unmyelinated axons of rat sciatic nerve

Summary Tannic acid in glutaraldehyde was used to stain microtubules in myelinated and unmyelinated axons of rat sciatic nerve. In the majority of areas the tannic acid failed to penetrate the unmyelinated axons whilst penetrating neighbouring myelinated axons, suggesting a difference in the ability of the two types of nerves to exclude tannic acid. Where tannic acid had penetrated the unmyelinated axons the 13 protofilament substructure and size of the microtubules appeared identical to those seen in the myelinated axons.     

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16-fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.     

The internal structure of axons from rat sciatic nerve

Summary Sciatic nerves from rats were examined electron microscopically following fixation in 4 % tannic acid in 2.5 % glutaraldehyde, which allowed demonstration of a filamentous network between the usual intra-axonal organelles. The network appears to consist of longitudinal 10 nm in diameter filaments and cross-linking filaments of about 6 nm diameter. Exposure to cold caused disruption of microtubules, but not the filaments, and incubation at 37°C following cold exposure resulted in reformation of the microtubules which again showed linking with the filaments. Exposure of the nerves to cold in the presence of D₂O did not cause disruption of the microtubules but there did appear to be some loss of the fine filaments. These findings suggest that the finer cross-linking filaments are of a different nature than the longitudinal 10 nm filaments, and that there is a dynamic relationship between these filaments and microtubules since the cross-linkages reappear following microtubule disruption and reformation.     

Ultracytochemical identification of catecholamine-containing vesicles in the ligated sciatic nerve of the rat. Comparison with sympathetic nerve terminals

Summary Using the fixation procedure of Tranzer, three kinds of granular vesicles were identified in certain unmyelinated fibres of rat sciatic nerves proximal to a ligature: (1) small vesicles (SGV: 30–60 nm in diameter), (2) large vesicles (LGV: 60–100nm in diameter), and (3) large elongated vesicles (LEV: 60–100nm in diameter). A comparative study concerning the distribution of these granular vesicles was carried out using a cytopharmacological method (reserpine) and employing different fixatives (aldehydes + OsO₄, or OsO₄ alone) in periarterial nerve plexus of the femoral artery, vas deferens and the pineal organ.Use of Tranzer's method allows preservation in almost all granular vesicles of a strongly electron-dense core, while with the other fixatives mainly small, eccentric dense cores occur in the vesicles. Two main features were observed in ligated sciatic nerves: (i) a clear increase in the number of LGV, and (ii) the presence of LEV, considered as a variety of LGV rather than a new population of granular vesicles. Reserpine caused the cores of SGV to disappear almost completely, while LGV and LEV remained only partly depleted. The original method combining Tranzer's fixation procedure with radioautography revealed radioautographic labelling only in the unmyelinated fibres of ligated sciatic nerves and mainly superimposed over SGV, LGV and LEV. It is suggested that (i) SGV, LGV and also LEV represent possible storage sites of catecholamines, and (ii) a local morphogenesis of SGV from the large vesicles occurs in ligated sympathetic nerve fibres.     

Detection of sodium channel distribution in rat sciatic nerve following lysophosphatidylcholine-induced demyelination

Summary In vivo application of lysophosphatidylcholine (LPC) to rat sciatic nerve induces impaired hind leg movement within 2 days which is recovered by 6 days. Segmental demyelination was seen at 2 days after LPC application, and remyelination had barely started in a few axons by 6 days. Using sodium channel-specific monoclonal antibodies and immunofluorescence microscopy, we observed altered distribution of sodium channels in demyelinated axons. Bright fluorescent labeling was found along the segmentally demyelinated axolemma at 6 days in contrast to the dim staining of the demyelinated nerve found at 2 days. In addition, radioimmunoassays detected an elevated number of antibody binding sites on sciatic nerve trunk from the sixth day. Our data provide the immunocytochemical evidence for the assumption that recruitment of sodium channels into demyelinated axolemma contributes to the recovery of function following axon demyelination by LPC.     

Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M-null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M-null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.     

Behavioral evaluation of regenerated rat sciatic nerve by a nanofibrous PHBV conduit filled with Schwann cells as artificial nerve graft

Abstract

The aim of this study is to develop a nanofibrous polymeric nerve conduit with Schwann cells (SCs) and to evaluate its efficiency on the promotion of functional and locomotive activities in rats. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the rats were monitored and evaluated by behavioral analyses such as toe out angle, toe spreading analysis, walking track analysis, extensor postural thrust, open-field analysis, swimming test and nociceptive function, four months post surgery. Four months post-operatively, the results from behavioral analyses demonstrated that in the grafted groups especially in the grafted group with SCs, the rat sciatic nerve trunk had been reconstructed with functional recovery such as walking, swimming and recovery of nociceptive function. This study proves the feasibility of artificial conduit with SCs for nerve regeneration by bridging a longer defect in the rat model.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve

BackgroundAging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most physiological indices of aging.ObjectivesThis work aimed to investigate the effect of CR on age-related changes in sciatic nerves.Materials and methodsThirty male albino rats aged 1 month were equally divided into three groups; Group I [control adult-ad libitum AL]: fed a regular diet and sacrificed at the age of 6 months, group II (aged-AL group): fed a regular diet AL and sacrificed at the age of 18 months, and group III (aged CR) fed a 40% calorie restricted diet and sacrificed at the age of 18 months. Rats were anesthetized and sciatic nerves were processed for light, electron microscope and morphometric studies. Oxidative stress in sciatic nerves was investigated by estimation of lipid perioxidation by product malondialdehyde (MDA) tissue level and antioxidant enzyme; superoxide dismutase activity (SOD).ResultsThe aged (AL) sciatic nerves appeared disorganized, with thick perineurium and increased collagen fibers associated with decreased g-ratio. Abnormal myelin forms were seen as outfolded myelin loops, thin denuded myelin, splitting of myelin into myelin figures and interlamellar vacuoles. Schwann cells revealed vacuolated cytoplasm. There was also significant increase in MDA level and a significant decrease in SOD activity in comparison to control adult (AL). Apparent structural and histomorphological improvement were noticed after CR in aged rats.ConclusionAging caused structural and biochemical alterations in sciatic nerves with alleviating effect of calorie restriction on such effects.     

Age-related variations of elements as compared among optic, radial, and sciatic nerves

To elucidate changes of peripheral nerves with aging, the authors studied age-related changes of element contents in the optic, radial, and sciatic nerves by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The contents of phosphorus and sulfur remained constant through ages 61 to 97 yr in three nerves, the optic, radial, and sciatic nerves. It was found that there were age-related differences in calcium content among the optic, radial, and sciatic nerves: The calcium content of the optic nerve increased progressively with aging; in the radial nerve, it was hardly changed with aging; in contrast, the calcium content of the sciatic nerve decreased gradually with aging. In addition, it was found that in the radial nerve there were moderate correlations between age and zinc or sodium content, whereas significant correlations between age and the content of silicon or iron were found in the sciatic nerve. Furthermore, there was a correlation between the silicon and iron contents in the sciatic nerves.     

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.     

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of¹²⁵I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8±0.4 mm/h) than in mouse sciatic nerves (5.4±0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0–2.5 h) with a reversal to normal rate 3–5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.I had the good opportunity of being a member of Professor Alan N. Davison' research team during 1971–1977. This research paper is dedicated to his retirement.     

大鼠坐骨神经挤压损伤导致脊髓单突触反射回返性抑制的长时程减弱

坐骨神经损伤是临床常见的周围神经疾病。神经损伤后再生肌肉和运动神经元会出现各种功能障碍,虽然其中一部分因素已被阐明,但多局限于受损神经局部,而对于再生后脊髓运动神经元的回返性抑制(recurrent inhibition,RI)通路的功能变化却很少被报道。本文研究大鼠短暂坐骨神经损伤后,恢复神经再支配(reinnervation)情况下,脊髓RI通路的功能变化。在正常或坐骨神经挤压(crush)受损后的成年大鼠上,通过刺激离断的脊髓背根(L5),在外侧腓肠肌-比目鱼肌(lateral gas-trocnemius-soleus,LG-S)神经或内侧腓肠肌(medial gastrocnemius,MG)神经记录单突触反射(monosynaptic reflex,MSR),并同时在另一神经给予条件性刺激,以检测LG-S和MG运动神经元间RI的变化。结果显示:(1)脊髓运动神经元的RI在坐骨神经挤压受损后即基本丢失(<5周),至损伤6周后部分恢复至正常的50%,并至少维持至损伤14周后;(2)一侧的坐骨神经损伤对对侧的RI没有影响;(3)外周神经损伤后,免疫组织化学方法显示脊髓运动神经元数目本身并不发生减少。以上...     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Pretreatment of rats with pulsed electromagnetic fields enhances regeneration of the sciatic nerve

Regeneration of the sciatic nerve was studied in rats pretreated in a pulsed electromagnetic field (PEMF). The rats were exposed between a pair of Helmholtz coils at a pulse repetition rate of 2 pps at a field density of 60 or 300 μT. The PEMF treatment was then discontinued. After an interval of recovery, regeneration of the sciatic nerve was initiated by a crush lesion. Regeneration of sensory fibers was measured by the “pinch test” after an additional 3–6 days. A variety of PEMF pretreatments including 4 h /day for 1–4 days or exposure for 15 min/day during 2 days resulted in an increased regeneration distance, measured 3 days after the crush lesion. This effect could be demonstrated even after a 14-day recovery period. In contrast, pretreatment for 4 h/day for 2 days at 60 μT did not affect the regeneration distance. The results showed that PEMF pretreatment conditioned the rat sciatic nerve in a manner similar to that which occurs after a crush lesion, which indicates that PEMF affects the neuronal cell body. However, the mechanism of this effect remains obscure. © 1993 Wiley-Liss, Inc.     

Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

Oxygen toxicity in the nervous tissue: Comparison of the antioxidant defense of rat brain and sciatic nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.