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 CNI 100 (TM) 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. (c) 2007 Elsevier Ltd. All rights reserved.

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons

Eugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na(+) channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications. eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Inside-out vein graft and inside-out artery graft in rat sciatic nerve repair

Although veins and arteries present similar wall structures, there are differences which may be relevant in peripheral nerve reconstruction. Inside-out vein grafts (IOVG) have been satisfactorily used to repair both motor and sensitive nerves. However, the inside-out artery graft (IOAG) is a new technique and not fully investigated. Our study presents comparative morphological data on nerve regeneration achieved with IOVG and IOAG in the repair of Wistar rat sciatic nerves. Jugular veins and aorta arteries were harvested from donor animals and used inside-out to bridge a 10-mm gap. Animals were sacrificed at 10 weeks to evaluate nerve regeneration. Both techniques presented great variability in nervous tissue, though some animals showed satisfactory results. Different intensities of scarring processes might have interfered with nerve regeneration. Although IOVG and IOAG techniques showed similar morphometric results, in general, IOVG presented a closer-to-normal nerve organization than IOAG.

Sciatic nerve regeneration in rats by a nerve conduit engineering with a membrane derived from natural latex

PURPOSE: To evaluate the capacity of natural latex membrane to accelerate and improve the regeneration quality of the of rat sciatic nerves. METHODS: Forty male adult Wistar rats were used, anesthetized and operated to cut the sciatic nerve and receive an autograft or a conduit made with a membrane derived from natural latex (Hevea brasiliensis). Four or eight weeks after surgery, to investigate motor nerve recovery, we analyzed the neurological function by walking pattern (footprints analysis and computerized treadmill), electrophysiological evaluation and histological analysis of regenerated nerve (autologous nerve graft or tissue cables between the nerve stumps), and anterior tibial and gastrocnemius muscles. RESULTS: All functional and morphological analysis showed that the rats transplanted with latex conduit had a better neurological recovery than those operated with autologous nerve: quality of footprints, performance on treadmill (p<0.01), electrophysiological response (p<0.05), and quality of histological aspects on neural regeneration. CONCLUSION: The data reported showed behavioral and functional recovery in rats implanted with latex conduit for sciatic nerve repair, supporting a complete morphological and physiological regeneration of the nerve.

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of(a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadonopin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.

Long term peripheral nerve regeneration using a novel PCL nerve conduit.

The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.

雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究

在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA 文库，利用特异引物筛选到Anntoxin 和AnSF 的cDNA 编码序列，前者的Gene Bank 登录号为FJ598043，后者还在等待分配登录号。Anntoxin 具有60 个氨基酸，是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，构建Anntoxin 的3D-NMR 溶液结构，证实Anntoxin 不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz 类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF 具有123 个氨基酸，在C 端具有和Calmadolin 同源的两个EF 手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。为了进行Anntoxin 的生物活性和结构分析，我们在体外成功表达了 Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析， rAnntoxin 和天然分离到的Anntoxin 生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin 是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX 或蛋白酶抑制剂K 分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin 也有一定的同源性。利用膜片钳技术分别检测Anntoxin 对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin 对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对 K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1 和 Kv4.2，Kv4.3，Anntoxin 对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin 的3D-NMR 溶液结构（NMR 号：PDB ID 2KCR， BMRB ID 16094），证实Anntoxin 具有典型的Kunitz 结构，由反向平行的 β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot 以及 ELISA 技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA 转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、 4.80 和2.02 微克/克鲜重，可以看出Anntoxin 在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin 有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin 对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50 分别为50，450，2500 和3000 微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin 对几类潜在天敌确实有较强的杀灭作用。为了检测AnSF 的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100 和500ng/ml，把AnSF 和hESC 共培养，发现在10~100 ng/ml 浓度时对hESC 的自我更新有支持作用；设计三个浓度梯度10、100 和500ng/ml，把AnSF 和rNSC 共培养，发现在 10ng/ml 时对rNSC 的自我更新有较强的支持作用。在超过500ng/ml 高浓度时，AnSF 对hESC 和rNSC 都有明显的细胞毒性作用，对rNSC 的毒性作用更明显。利用RT-PCR 技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF 只在皮肤中有少量表达。这表明AnSF 可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF 的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF 是维持华西雨蛙皮肤内环境稳定的重要物质。

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

The analgesic effect of crotoxin on neuropathic pain is mediated by central muscarinic receptors and 5-lipoxygenase-derived mediators

Crotoxin (CTX). a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg. i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). on the other hand. indomethacin (4 mg/kg, i.v.). naloxone (1 mg/kg, i.p.). and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of alpha-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin. (C) 2008 Elsevier B.V. All rights reserved.

ESTUDO DO EFEITO DE GANGLIOSIDEOS EXOGENOS NA REGENERACAO NERVOSA APOS COMPRESSAO E NEURORRAFIA NO NERVO CIATICO DO RATO

The authors studied the possible inductive effect of exogenous gangliosides administrated intra-peritoneal and intra-muscular, in 33 rats, after compression and sutures of sciatic nerve. In the animals suffering compression of the sciatic nerve only, the myelin sheath was preserved with good regeneration of the nerve. There was no statistics difference between the two groups with or without the administration of gangliosides. In the group of rats where section of the sciatic nerve and suture were performed, neuroma of amputation as well as dense cicatricial tissue with inflammatory reaction of foreign body type was noted around the sutures. In conclusion, the formation of neuroma of amputation associated with severe inflammatory reaction had an important role in determining the regeneration of the nerve.

Use of fibrin glue derived from snake venom in testicular biopsy of rams

Sequelae due to testicular biopsy such as hemorrhage, adhesion and fibrosis may be limiting factors to the use of this surgical procedure. Fibrin glue (FG) derived from snake venom was used to minimize these sequelae, as well as to evaluate its healing property in tunica vaginalis and scrotal skin of rams. Applicability of fibrin glue derived from snake venom was tested in different tissues of other animals such as in sciatic nerve and colon of rats and skin of rabbits. In the present study, 30 healthy adult rams were used. They were divided into 3 groups of 10 animals each as follows: G1: fibrin glue group (application of fibrin glue on puncture sites and skin incisions after bilateral testicular biopsy with a Tru-Cut needle); G2: swab/nylon group (hemostasis by compression with a swab on puncture sites and skin suturing with nylon after biopsy) and G3: control group (the animals were not subjected either to biopsy or to surgery). On the 20th day after biopsy, the presence of adhesion strands between the sites of skin incision and testicle was evaluated by palpation Adhesion strands were found in three testicles (15%) in G1 and in two testicles (10%) in G2. One hundred days after biopsy, orchiectomy was carried out and the material collected was assessed for subcutaneous (SC) and/or tunica vaginalis adhesions. G3 did not present any abnormality. Groups G1 and G2 presented four testicles each (20%) with adhesion between the tunics at biopsy site. On the other hand, subcutaneous adhesions were found once (5%) in G1 and three times (15%) in G2. Fibrin glue showed to be of easy application, required short postoperative monitoring, presented fast and good-quality healing property and tended to reduce formation of subcutaneous adhesion.

Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?

Painful peripheral neuropathies are precipitated by nerve injury from disease or trauma. All such injuries will be accompanied by an inflammatory reaction, a neuritis, that will mobilize the immune system. The role of the inflammation itself is difficult to determine in the presence of structural damage to the nerve. A method has been devised to produce a focal neuritis in the rat sciatic nerve that involves no more than trivial structural damage to the nerve. This experimental focal neuritis produces neuropathic pain sensations (heat- and mechano-hyperalgesia, and cold- and mechano-allodynia) in the ipsilateral hind paw. The abnormal pain sensations begin in 1–2 days and last for 4–6 days, with a subsequent return to normal. These results suggest that there is a neuroimmune interaction that occurs at the outset of nerve injury (and perhaps episodically over time in slow developing conditions like diabetic neuropathy) that produces neuropathic pain. The short duration of the phenomena suggest that they may prime the system for more slowly developing mechanisms of abnormal pain (e.g., ectopic discharge in axotomized primary afferent neurons) that underlie the chronic phase of painful neuropathy.