肌电图检查的临床应用意义 肌电图学(electromyography, EMG)是研究神经和肌肉细胞电活动的科学,而肌电图(electromyogram,临床上习惯也用EMG作为其简称)是指肌肉在安静和收缩状态下的电生理特性的记录,一般包括广义和狭义两层意义:狭义EMG也称为同心圆针或常规EMG;而广义EMG则包括神经电图或称为神经传导速度、重复神经电刺激、各种反射、单纤维肌电图、巨肌电图、扫描肌电图、表面肌电图、诱发电位肌电图及运动单位计数等。临床上常常根据病人的临床表现和印象诊断相应调整和决定检查的具体项目。自1961年Pavia(意大利)第一届国际肌电图学会议后,经Jun Kimura等学者的研究与开拓,发现EMG检查在神经肌肉疾病的诊断、疗效评估、预后评价方面有着重要价值。国外学者甚至提出——EMG是神经系统检查的延伸。 EMG检查的适应症及意义: 一、适应症:前角细胞以下包括前角细胞的病变 二、临床意义: 1.发现临床下病灶或易被忽略的病变 运动神经元病的早期诊断深部肌肉萎缩和轻瘫 2.诊断和鉴别诊断 神经源性损害?/肌源性损害?/还是神经肌肉接头病变? 髓鞘损害?/轴索损害? 3.提供临床定位诊断的依据 上运动神经元病变?/下运动神经元病变? 广泛性病变?/节段性病变? 单神经损害?/多神经损害? 神经元损害?/神经根损害?/神经干损害?/神经末梢损害?等等。 4.判断病情及预后评价 5.疗效评估。
ORIGINAL ARTICLEYear : 2010 | Volume : 58 | Issue : 4 | Page : 530-536Protective effects of the calcium-channel blocker flunarizine on crush injury of sciatic nerves in a rat modelJian-Hua Su1, Yu-Fang Chen2, Jin-Rong Tang1, Le Wu3, Ping Zhang4, Long-Bin Yu5, Qi Niu1, Hang Xiao3Date of Acceptance10-Jun-2010Date of Web Publication24-Aug-2010Correspondence Address:Jin-Rong TangDepartment of Neurology, The First Affiliated Hospital of Nanjing Medical University, Nanjing - 210 029 Chinadoi:10.4103/0028-3886.68665PMID:20739787AbstractBackground : Neural damage can be mitigated by calcium-channel blockers (CCBs). However, the mechanism of action of CCBs is not yet fully understood. Objective : To investigate the mechanism of action and efficacy of CCB, flunarizine in restoring neural function after crush injury to the nerves Materials and Methods : The sciatic nerves of rats were crushed using pincers to establish the model for crush injury. Two hundred and eighty-eight Sprague-Dawley (SD) rats were randomly divided into sham-operated, saline, and low-dose flunarizine and high-dose flunarizine (FI and FII) groups. The expression of the protein c-fos in the dorsal root ganglia (DRG) after crush injury to the sciatic nerves was investigated by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The effect of flunarizine on c-fos expression and its efficacy in restoring neural function was evaluated. Results : The c-fos messenger ribonucleic acid (mRNA) and protein expression in FI and FII groups was significantly lower than in the saline group and was the least in the FII group. Nerve-conduction velocity was increased in the order of: saline < FI< FII< sham-operated. There was no significant difference in the nerve-conduction velocity in the sham-operated and FII groups (P>.05). Conclusions : When administered after crush injury to peripheral nerves, flunarizine may protect neurons with lesions from further damage and improve neural function by downregulating c-fos expression.Keywords:c-fos, crush injury, dorsal root ganglion, flunarizine, nerve conduction velocity, sciatic nerveIntroductionThe protein c-fos is widely expressed in neurons and acts as the third messenger in the signal transduction pathway in neurons. Recent studies have investigated its role in the transduction of harmful signals. Dragunow and Faull [1] reported that c-fos is located basally in neurons and that its expression can increase after behavioral stress. Kaczmarek and Nikofajew [2] reported that increase in c-fos messenger ribonucleic acid (mRNA) and/or protein expression is caused by the action of neurotransmitters on membrane receptors which, in turn, is induced by physiological stimuli. According to Shortland and Molander [3] stimulation of A-beta afferents induce expression of c-fos in postsynaptic cells. In addition, c-fos is expressed in the axons involved in the conduction of nociceptive stimulus soon after nerve trunk injuries. Other studies have proposed that nerve lesions caused due to various stimuli can be investigated by detecting the loci and level of c-fos expression. [4],[5],[6],[7],[8] Various factors, such as ischemia, inflammation, and trauma, can cause calcium influx, which in turn can damage neurons and axons. Neural damage thus induced can be mitigated by administering calcium-channel blockers (CCBs). [9],[10] However, the mechanism of action of CCBs is not yet fully understood. Calcium influx can also be induced by the overstimulation of N-methyl-d-aspartate (NMDA) receptor. [11],[12] We investigated the mechanism of the protective effect of CCB after crush injury to peripheral nerves, expression of c-fos protein at an early stage of crush injury, pathological changes of sciatic nerve trunks, and the nerve conduction velocities (NCVs) of sciatic nerves at week 4 after sciatic nerve crush injury in rats. The effect of a CCB, flunarizine was also examined on c-fos expression and repair of neural lesions.Materials and MethodsSciatic nerve injury in rats We obtained 256 Sprague-Dawley (SD) rats that were of the specific-pathogen-free (SPF) grade (128 males and 128 females; body weight: 178-224 g) from the Animal Center of Nanjing Medical University, Nanjing, China. After 12 h fast the rats were randomly divided into sham-operated, saline, low-dose flunarizine (FI), and high-dose flunarizine (FII) groups. Each group included 64 rats (32 males and 32 females). All study procedures were performed in accordance with the animal care guidelines followed at the Nanjing Medical University, which conform to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised, 1985). The rats were fixed onto rat plates after they were anesthetized with intraperitoneally (IP) administration of 3% pentobarbital (10 mL/kg). An incision was made in the right hind limb and the sciatic nerve was exposed. In the sham-operated group, the incision was closed after exposing the sciatic nerve. The sciatic nerves of the rats in other groups were clipped with artery forceps (surgical hemostatic forceps, straight, 14 cm Cr, Zhangjiagang Shuangyin Apparatus Co., Ltd., Zhangjiagang, China) by applying pressure equal to half the body weight of the rats with accurate electronic scales (OCS-XZ-GSC, Nanjing Lianyu Measuring Equipment Co., Ltd., Nanjing, China) for 30 s; the pressure was then released and the incisions were closed. The rats were administered antibiotics to prevent infections. The rats in the low-dose group were given 1 mg/(kgd) of flunarizine [It was equivalent to 10 mL/(kgd) solution after dilution; batch number 030118687; Xi'an-Janssen Pharmaceutical Company, Xi'an China] and the high-dose group received 2 mg/(kgd) intraperitoneally. The rats in the saline group were administered an equivalent volume of saline.Separation of dorsal root ganglia and sciatic nerve trunksThe rats were killed and their dorsal root ganglia (DRG) and sciatic nerve trunks (which comprise the distal tibial nerve) were immediately separated by the Li's method [13] . The lumbar and sacral segments of the spinal cord and cauda equina were removed. The section was made along the midline of the body. The spinal segments, DRG, neural roots, and the sciatic nerve trunks were placed in culture dishes containing oxygen-enriched, saturated Dulbecco's modified Eagle's medium (DMEM; pH 7.4; osmotic pressure, 340 mOsm/L). The DRG, nerve roots (anterior and posterior roots), and sciatic nerve trunks were removed from the neural canal of the rats. The membrane of the peripheral connective tissue, which connects to the nerve trunk, was removed under a stereoscopic microscope, using fine corneal scissors and wire forceps.Detection of c-fos mRNA in DRG cells by using reverse transcription-polymerase chain reactionEight rats from each group were culled after administering flunarizine at 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and their spinal cord, DRG, neural root (anterior and posterior roots), and sciatic nerve trunk were separated. RNA was extracted from the separated DRG and reverse transcribed using SuperScript II reverse transcriptase, according to the manufacturer's instructions, to make up a total reaction volume of 20 μL. First-strand complementary RNA was synthesized from 4 μg of total RNA by using 0.5 μg of oligo (dT) primers, 1Χ first-strand buffer, 0.01 M dithiothreitol (DTT), 0.5 mM deoxyribonucleotide (dNTP) mix, and 200 U of SuperScript II at 42°C for 50 min. The reaction was stopped by heating at 70°C for 15 min. Each sample was amplified by performing 35 cycles of polymerase chain reaction (PCR) using oligonucleotide primers: c-fos primer 5′ATGATGTTCT-CGGGTTTCAA-3′ (forward) and 5′-TGACATGGTCTTCACCACTC-3′ (reverse), amplifies a 348-bp fragment; β-actin primer 5′-CCACGAGAAGATGACCCAGAT-3′, 677-bp fragment (Beijing Sanboyuan Biotechnology Limited-liability Company, Beijing China). The amplification conditions were as follows: Initial denaturation was performed at 95°C for 2 min, followed by 35 cycles of denaturation (at 95°C for 1 min), annealing (at 55.5°C for c-fos/56°C for c-jun) for 1 min, extension (at 72°C for 1 min), and final elongation (at 72°C for 2 min). The amplified products were resolved on agarose (1%) by performing homeothermic gel electrophoresis at 80 v for 30 min. The bands were excised and eluted from the gel, purified, precipitated overnight with ethanol, and sequenced. The electrophoresis results were observed under an ultraviolet lamp and a density scan of the positive bands was performed. Then, the refractive index (RI) of c-fos mRNA was calculated using the formula RI = c-fos mRNA density/β-actin density Χ 100%.Detection of c-fos protein in DRG by Western blotEight rats from each group were culled after administering flunarizine at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h, and their spinal cord, DRG, the connected neural root (anterior and posterior roots), and sciatic nerve trunk were isolated. The isolated DRG were infused with 4% paraform, and dehydration and paraffin embedding were performed using routine methods. The specimens were cut into 50-μm sections, and avidin-biotin peroxidase complex (1:150; ABC; Sigma, USA) immunohistochemical stain was used to detect c-fos expression in DRG. Then, the number of c-fos-positive cells in 5 microscopic fields Χ 200 LM was counted to determine the average number of cells in each field.Pathological examination The rats in each group were sacrificed at week 4 after crush. The DRGs and sciatic nerves were separated according to the method previously described. The sciatic nerve trunk was stained by Weil's medullary sheath staining method. Pathological examination of the sciatic nerves was performed under a light microscope.Nerve conduction velocities (NCV) of sciatic nervesEight rats from each group were killed at week 4 after the crush injury. Then a 6-cm length of sciatic nerve (which comprise the distal tibial nerve) trunk was separated. The nerve conduction velocities (NCVs) of the sciatic nerves were detected at 2 min, 3 min, and 4 min using D95 Super NCV lab determinator (Medical Electrons Institute of Academy of Jiangsu Biomedical Engineering, Nanjing, China). The characteristics of each stimulus impulse were as follows: 0.04 ms; 2.7 v; and scanner speed, 50000 mm/s. The recording electrodes were placed 3.5 cm apart on every sciatic nerve trunk and the temporal changes in the action potential were recorded.Statistical analysisStatistical analysis was performed using Statistical Package for the Social Sciences (SPSS; version 11.5; Bizinsight, Beijing, China). P<.05 was considered to be significant. Intergroup data were compared using analysis of variance (ANOVA). The quantities of c-fos mRNA and protein were consistent with normal distributions and were analyzed using Student-Newman-Keul's (SNK) test.Resultsc-fos mRNA expression in DRGThe levels of c-fos expression in the sham-operated group were low at all the post-injury time points of measurement. In the saline, FI, and FII groups, the baseline levels (at 0 h) of c-fos mRNA expression were lower than that at the 30-min time point (P<.01) and were the highest at the 1-h time point (P<.01), after which they tended to decrease. However, in all these three groups, the expression levels of c-fos mRNA at 2 h after the injury were higher than those at baseline (P<.01). The c-fos mRNA expression levels in the other groups at 30 min, 1 h, and 2 h after the crush injury were obviously higher than those in the sham-operated group (P<.01). The c-fos mRNA expression levels in the FI and FII groups at 30 min, 1 h, and 2 h after the crush injury were significantly lower than those in the saline group (P<.01). The c-fos mRNA expression level in the FII group was significantly lower than that in the FI group (P<.01) [Figure 1],[Table 1]. Table 1 :c-fos mRNA Click here to viewFigure 1 :(a) c-fos mRNA in sham-operated group (M marker, N negative)Figure 1b: c-fos mRNA in saline group (M marker, N negative)Figure 1c: c-fos mRNA in FI group (M marker, N negative).Figure 1d: c-fos mRNA in FII group (M marker, N negative)Figure 1e: -actin (M marker, N negative)Click here to viewc-fos protein expression in DRGThe levels of c-fos expression in the sham-operated group were low at all the post-injury time points of measurement. In the saline, FI, and FII groups, c-fos expression level (at 0 h) tended to increase after the 1-h time point (P<.01) and reached peak expression levels at the 2-h time point (P<.01). The c-fos expression level started to decrease after the 2-h time point, but remained higher than the baseline level even 24 h after the injury (P<.01). The c-fos protein expression levels in the other groups at 1 h, 2 h, 4 h, and 24 h after the crush injury were obviously higher that those in the sham-operated group (P<.01). The c-fos protein expression levels in the FI and FII groups at 1 h, 2 h, 4 h, and 24 h after the crush injury were significantly lower than those in the saline group (P<.01). The c-fos protein expression level in the FII group was significantly lower than that in the FI group (P<.01) [Figure 2],[Table 2]. Table 2 :c-fos protein Click here to viewFigure 2Click here to viewPathological changes of sciatic nerve trunks detected by light microscopyThere was extensive and severe myelinoclasis and vacuolar degeneration of the sciatic nerve trunk in the saline group. There was segmental and mild myelinoclasis and a few instances of vacuolar degeneration of the sciatic nerve trunk in the FI and FII groups; this was milder in the FII group. There were no pathological changes in the sham-operated group [Figure 3]. Figure 3 :(a) Pathological changes of sciatic nerve trunks in sham-operated group (stained by Weil's medullary sheath staining method)Figure 3b: Pathological changes of sciatic nerve trunks in saline group (stained by Weil's medullary sheath staining method)Figure 3c: Pathological changes of sciatic nerve trunks in FI group. (stained by Weil's medullary sheath staining method)Figure 3d: Pathological changes of sciatic nerve trunks in FII group. (stained by Weil's medullary sheath staining method)Click here to viewNCVs of sciatic nervesAt week 4 after the crush injury, the NCVs of the sciatic nerves in the sham-operated and FII groups were greater than that in the FI and saline groups (P<.01). There was no difference between the sham-operated and FII groups or between in the saline and the FI groups (P>.05) [Table 3]. Table 3 :NCVs of sciatic nerves Click here to viewDiscussionAs has been explained by Narita, c-fos expression in the nerve trunk is the response of axons to nociceptive stimuli. [4] The results of our study showed that the c-fos expression in the saline and flunarizine groups significantly increased at 30 min after the crush injury of the sciatic nerves in the SD rats. These observations are similar to Curran's results, where maximal levels of c-fos mRNA expression were detected at 30 min after treatment with growth factors, c-fos protein expression was high for about 2 h after the injury, and cell morphology remained normal at the latter time point. [14] It was reported by Morano that a single toe pinch in rats produced nuclei- and condition-specific neuronal responses in the anterior region of the bed nucleus of the stria terminalis. Particularly, acute noxious stimulation increased c-fos expression in the dorsal medial and fusiform nuclei of the bed nucleus. Chronic neuropathic pain induced by chronic constriction injury of the sciatic nerves led to decrease in the number of c-fos-positive cells after acute mechanical stimulation in the dorsal medial and fusiform nuclei and increased c-fos immunoreactivity in the ventral medial aspect of the bed nucleus of the stria terminalis. [15] In our study, the c-fos expression in the FI and FII groups was less than that in the saline group (the difference was more significant in the FII (high-dose) group (P<.01). It was reported that pretreatment with l-type CCB - capsaicin - completely prevented mechanical hyperalgesia induced by disc compression. Tang et al. [16] reported that the obvious expression of c-fos-like immunoreactive neurons in the dorsal horn of the spinal cord, which they brought about by direct compression of the L5 nerve root, was markedly decreased by pretreatment with capsaicin. In addition, pretreatment with an l-type CCB suppressed the haloperidol-induced c-fos expression throughout the neostriatum and the nucleus accumbens when evaluated 2 h after the injection of the CCB. [17] However, c-fos protein expression observed only in the lateral part of the neostriatum at 5 h after the injection of haloperidol in rats pretreated with l-type CCB was higher than that in rats pretreated with the vehicle alone. In addition, pretreatment with the l-type CCB prolonged the duration of haloperidol-induced catalepsy in rats. Infusion of the l-type CCB directly into the neostriatum mimicked the patterns of changes caused by haloperidol-induced c-fos expression. Ca ++ acting as a second messenger stimulated the expression of c-fos, [5] and flunarizine downregulated the expression of c-fos by blocking the calcium influx. [4],[5] In our study, the pathological changes of the sciatic nerve trunk in the FI and FII groups [especially, in the FII (high-dose) group] were milder than those in the saline group at week 4 after the crush injury. The NCVs of the sciatic nerves in the FII group were greater than the NCVs of the sciatic nerves in the saline and FI groups (P<.01). The spaces between the toes of the rats in the FI and FII groups were significantly greater than that seen in the rats in the saline group (P<.01). Patro et al. [18] reported that flunarizine administration markedly reduced the extent of DRG neuron loss. In their experiment, PRTS was 89 and 95.7% in the sciatic nerve crush (SNC) group and the SNC + flunarizine group, respectively. Similarly, the data on 2-4-toe spread suggested a protective action of flunarizine. The PRTS was 94.5 and 99% at the end of the experiment in the SNC and the SNC + flunarizine groups, respectively. The treatment improved the percentage relative toe-spread also significantly greater than the untreated injured rats. The dosages of flunarizine in this experiment was higher than that used in Patro et al.'s [18] experiment, but the toe spaces of the saline, FI, and FII groups (which was 68.03%, 87.08% and 96.95%, respectively, of that in the sham-operated group) was less than that found by Patro et al. [18] It might be that tested toe spaces was different. Why did not flunarizine completely inhibit the pathological lesions caused by crush injury of the sciatic nerves in rats? Was the development of lesions influenced by other ion channels? Mert et al. found that the slow and fast K + channels and slow Na + currents affect the membrane potential and depolarization of the action potential of neurons. Myelin damage, even if it is minimal, might markedly affect subsequent impulse generation and the pattern of action-potential activity. [19] Varejao et al. reported that there was good correlation between sciatic functional index and toe-out angle measurements in predicting functional recovery. [20] Luis et al. [21] proposed that the combined functional and morphological analyses should be performed in experiments aimed at predicting functional recovery. Our results suggest that the expression of c-fos in the early stage after crush injury could affect subsequent sciatic function. Higher c-fos expression, low NCV, and administration of the CCB flunarizine may lead to the downregulation of c-fos expression soon after crush injury, thereby decreasing the pathological damage and increasing NCVs by blocking the calcium influx. The results confirmed the opinion of Yang and Averbeck that calcium influx can be induced by various factors and can impair neurocyte function. [7],[10] The findings reported by Matthews indicated that activity of the voltage-dependent Ca ++ channel was important for sustaining the release of neurotransmitters and excitability of neurons and that early use of flunarizine might protect against the loss of important functions. [22] Recently, Galtrey and Fawcett [23] opined that the final degree of functional recovery achieved was associated with retrograde axonal regeneration. The effects of retrograde axonal regeneration on neuronal function are most clearly evaluated by skilled paw-reaching and grip-strength tests. The lesion model and functional tests would be useful in testing therapeutic strategies for the effects of inappropriate axon regeneration following peripheral nerve injury in humans. In conclusion the overexpression of c-fos soon after crush injury to the peripheral nerves can induce pathological damage and adversely affect neural functions. CCBs block calcium influx into neurons, which in turn may inhibit expression of c-fos, thereby mitigating neuronal damage and improving neural functions.AcknowledgmentThis work was supported by a grant from the Medical Research Council [grant number: Natural Science of Jiangsu Province BK2001116, Item of Changzhou Board of Health 2002-202-17 and 2004-182-01, Item of Jintan Science and Technology Bureau 2002-28-25 ]. References1.Dragunow M, Faull R. The use of c-fos as a metabolic marker in neuronal pathway tracing. J Neurosci Methods 1989;29:261-5. 2.Kaczmarek L, Nikofajew E. c-fos protooncogene expression and neuronal plasticity. Acta Neurobiol Exp (Wars) 1990;50:173-9. 3.Shortland P, Molander C. Alterations in the distribution of stimulus-evoked c-fos in the spinal cord neonatal peripheral nerve injury in the rat. Brain Res Dev Brain Res 2000 ;119:243-50. 4.Narita M, Ozaki S, Narita M, Ise Y, Yajima Y, Suzuki T. Change in the expression of c-fos in the rat brain following sciatic nerve legation. Neurosci Lett 2003;352:231-3. 5.Sun M, Song XS, Gao JG, Wang S. Changes of behavior and CGRP: c-fos in habenula after chronic compression of DRG in rats. Heilongjiang Med J (Chin) 2002;26:243-5. 6.Kominato Y, Tachibana T, Dai Y, Tsujino H, Maruo S, Noguchi K. Change in phosphorylation of ERK and fos expression in dorsal horn neurons following noxious stimulation in a rat model of neuritis of the nerve root. Brain Res 2003;28:89-97. 7.Yang DZ, Wang KZ, Chen JC, Wang D, Liu HT, Xu L, et al. The expression of c-fos and transmitter calcitonin gene-related peptide in the chronic compressive injury of the nerve root. Zhonghua Wai Ke Za Zhi 2004;42:1236-9. 8.Yang Z, Rao Z, Qiu J. Subdiaphragmatic vagotomy inhibits fos expression in the medullary visceral zone after intraperitoneal administration of lipopolysaccharide. Acta Anatomica Sinica 2000; 31:97-101. 9.Huang CS, Song JH, Nagata K, Yeh JZ, Narahashi T. Effects of the neuroprotective agent liluzole on the high voltage-activated calcium channels of rat dorsal root ganglion neurons. J Pharmacol Exp Ther 1997;282:1280-90. 10.Averbeck B, Izydorczyk I, Kress M. Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat. Neuroscience 2000;98:135-40. 11.MacDonald JF, Xiong ZG, Jackson MF. Paradox of Ca2+ signaling, cell death and stroke. Trends Neurosci 2006;29:75-81. 12.Nicholls DG, Johnson-Cadwell L, Vesce S, Jekabsons M, Yadava N. Bioenergetics of mitochondria in cultured neurons and their role in glutamate excitotoxicity. J Neurosci Res 2007;85:3206-12. 13.Li S, CuI CD, Guan BC, Li ZW. Effect of 5-HT on the GABA membrane current of dorsal root ganglia. J Binzhou Med Coll (Chin) 1998;21:318-20. 14.Curran T, Bravo R, Muller R. Transient induction of c-fos and c-myc in an immediate consequence of growth factor stimulation. Cancer Surv 1985;4:655-81. 15.Morano TJ, Bailey NJ, Cahill CM, Dumont EC. Nuclei-and condition-specific responses to pain in the bed nucleus of the stria terminalis. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:643-50. 16.Tang JG, Chen HS, Yuan W, Hou S, Wang X, Zhou X. The role of capsaicin-sensitive primary afferents in experimental sciatica induced by disc herniation in rats. Spine (Phila Pa 1976) 2008;33:163-8. 17.Lee J, Rushlow WJ, Rajakumar N. l-type calcium channel blockade on haloperidol-induced c-fos expression in the striatum. Neuroscience 2007;149:602-16. 18.Patro IK, Chattopadhyay M, Patro N. Flunarizine enhances functional recovery following sciatic nerve crush lesion in rats. Neurosci Lett 1999;263:97-100. 19.Mert T, Gunay I, Polat S. Alterations in conduction characteristics of crushed peripheral nerves. Restor Neurosci 2005;23:347-54. 20.Varejγo AS, Cabrita AM, Geuna S, Melo-Pinto P, Filipe VM, Gramsbergen A, et al. Toe out angle: a functional index for the evaluation of sciatic nerve recovery in the rat model. Exp Neurol 2003;183:695-99. 21.Luνs AL, Amado S, Geuna S, Rodrigues JM, Simυes MJ, Santos JD, et al. Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp. J Neurosci Methods 2007;163:92-104. 22.Matthews EA, Dickenson AH. Effects spinally delivered N-and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy. Pain 2001;92:235-46. 23.Galtrey CM, Fawcett JW. Characterization of tests of functional recovery after median and ulnar nerve injury and repair in the rat forelimb. J Peripher Nerv Syst 2007;12:11-27. Figures [Figure 1], [Figure 2], [Figure 3] Tables [Table 1], [Table 2], [Table 3] google_protectAndRun("ads_core.google_render_ad", google_handleError, google_render_ad);
ORIGINAL ARTICLEYear : 2009 | Volume : 57 | Issue : 4 | Page : 387-394Expression of VEGF and neural repair after alprostadil treatment in a rat model of sciatic nerve crush injuryJinrong Tang1, Ye Hua1, Jianhua Su1, Ping Zhang1, Xuejiang Zhu2, Le Wu3, Qi Niu1, Hang Xiao3, Xinsheng Ding11Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210 029, China2Department of Physiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210 029, China3Department of Neurotoxicology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210 029, ChinaDate of Acceptance25-Jul-2009Date of Web Publication10-Sep-2009Source of Support: None, Conflict of Interest: NoneDOI:10.4103/0028-3886.55583PMID:19770537AbstractBackground: Vasoactive drug alprostadil improves microcirculation and can be effective in treating disorders of peripheral nerves. Vascular endothelial growth factor (VEGF) has been shown to have protective action in cerebral ischemia, disorders of spinal cord, and also peripheral nerves. However, the mechanism of action of VEGF in peripheral nerve injuries is uncertain. Objectives: To study the effect of application of alprostadil on the pathological and functional repair of crush nerve injuries and also the expression of VEGF. Materials and Methods: Rat sciatic nerves were crushed by pincers to establish the model of crush injury. All of the 400 sprague dawley (SD) rats were randomly divided into: Control; saline; saline + VEGF-antibody; alprostadil; and alprostadil + VEGF antibody groups. The SPSS 11.5 software was used for statistical analysis. The expression of VEGF in dorsal root ganglia (DRGs), following crush injury to sciatic nerves, was studied by reverse transcribed-polymerase chain reaction (RT-PCR), immunohistochemistry, electromicroscope, and electrophysiology. The effects of alprostadil on expression of VEGF, repair of neural pathology, and recovery of neural function were also evaluated. Results: We found that VEGF messenger ribonucleic acid (mRNA) was significantly increased in alprostadil and alprostadil + VEGF-antibody groups, compared to the saline and saline + VEGF antibody groups. The number of VEGF-positive neurons was significantly increased in the alprostadil group, compared to the saline, saline + VEGF antibody, and alprostadil + VEGF antibody groups. Besides, addition of this drug also caused less pathological changes in DRGs, better improvement of nerve conduction velocities of sciatic nerves, and more increase of toe spaces of right hind limbs of rats. Conclusions: The vasoactive agent alprostadil may reduce the pathological lesion of peripheral nerves and improve the rehabilitation of the neural function, which may relate to upregulation of the expression of VEGF, following crush injury to the peripheral nerves.Keywords:Crush injury, dorsal root ganglion, sciatic nerve, vasoactive agent, vascular endothelium growth factorHow to cite this article:Tang J, Hua Y, Su J, Zhang P, Zhu X, Wu L, Niu Q, Xiao H, Ding X. Expression of VEGF and neural repair after alprostadil treatment in a rat model of sciatic nerve crush injury. Neurol India 2009;57:387-94How to cite this URL:Tang J, Hua Y, Su J, Zhang P, Zhu X, Wu L, Niu Q, Xiao H, Ding X. Expression of VEGF and neural repair after alprostadil treatment in a rat model of sciatic nerve crush injury. Neurol India [serial online] 2009 [cited2009 Dec 17];57:387-94. Available from:http://www.neurologyindia.com/text.asp?2009/57/4/387/55583IntroductionCrush injury to peripheral nerves can adversely affect neural microcirculation and capillary occlusion.[1] Consequently, ischemia and oxygen deficiency in nerves, destruction of the blood-nerve barrier, neural edema, disorder of the neural internal environment, and neural dystrophy appear, and can result in dysfunction of neural conduction. It has been reported by clinical and experimental studies that vasoactive treatment can alleviate the effects of lesions in peripheral nerves. [2],[3] Alprostadil is an effective agent for treating disorders of peripheral nerves. [4],[5],[6],[7] It is known that the vascular endothelial growth factor (VEGF) can protect against neuronal lesions. However, the changes in VEGF following crush injury to peripheral nerves, and the mechanisms of vasoactive treatment are incompletely understood. In order to explore the protective mechanisms involved in vasoactive treatment on peripheral nerves after injury, the sciatic nerves of rats were crushed, and the effect of the vasoactive agent, alprostadil, on expression of VEGF was assessed. Materials and MethodsSciatic nerve injury in ratsAfter fasting for 12 hours, 400 sprague dawley (SD) rats (200 males and 200 females, SPF grade, body weight 180-220g, Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into: Control; saline; saline + VEGF antibody; alprostadil; and alprostadil + VEGF antibody groups. Each group included 80 rats (40 males and 40 females). All procedures were performed in accordance with the animal care guidelines of the Nanjing Medical University, which conform to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised, 1985). The rats were fixed onto rat plates after they were anesthetized with 10ml/kg of 3% pentobarbital via intraperitoneal (ip) injection. An incision was made in the right hind limb to expose the sciatic nerve. [8] In the control group, the incision was closed after exposure of the sciatic nerve. The sciatic nerves of the rats in other groups were clipped using pressure equal to half of the body weight for 30s, the pressure was then released and the incisions were closed. Routine antibiotics were given to prevent infections. The rats in the alprostadil (Beijing Taide, batch number 2044T, Beijing, China) and VEGF antibody (Beijing Zhongsan Jinqiao Biotechnology Ltd., Beijing, China) groups were administered drugs (4 μg/kg/d, ip) and rabbit IgG polyclonal VEGF antibody (diluted with saline to 1:100, 2ml/d, ip), and an equivalent volume of saline (ip) was given to the rats in the saline group. Separation of dorsal root ganglia and sciatic nerve trunksThe dorsal root ganglia (DRGs) and sciatic nerve trunks were separated immediately after the rats were sacrificed. The ribs were sheared along both sides of the spine, and the spine's thoracic and lumbar segments were removed. The section was cut along the median line, the spine segments, DRGs, neural roots and the sciatic nerve trunks were placed in culture dishes containing oxygen-enriched, saturated Dulbecco's Modified Eagle's Medium (DMEM), pH 7.4, the osmotic pressure was 340mOsm/L. DRGs, nerve roots (anterior and posterior roots) and the sciatic nerve trunks were removed from the neural canal. The membrane of the peripheral connective tissue, which connects to the nerve trunk, was removed under a stereoscopic microscope using fine corneal scissors, wire forceps and scissors.Detection of vascular endothelial growth factor mRNA in dorsal root ganglia cellsThe DRGs were taken at time points including: 0, 3, 6, 12, 24, 48, 72, and 96 hours, as well as at day seven after crushing the sciatic nerves. Reverse transcribed-polymerase chain reaction (RT-PCR) was performed as follows (reverse transcribed kit from Promega, Madison, WI, TRIzol from Invitrogen, Carlsbad, CA):Total ribonucleic acid (RNA) was extracted from DRGs according to the manufacturer's instructions. The extracted RNA was incubated at 70°C for 10min and then placed on ice. The oligonucleotide primers for VEGF 165 were 5-GAAGTGGTGAAGTTCATGGATGTC-39(forward) and 5-CGATCGTTCTGTATCAGTCTTT CC-3(reverse), and amplified a 541 bp fragment; and for β-actin the primers were 5-CGCTGCGCTGGTCGTCGACA-3(forward) and 5-GTCACGCACGATTTCCCGCT-3(reverse), amplified a 619 bp fragment (Shanghai Shenggong Biotechnology limited-liability company, Shanghai, China). Amplification conditions consisted of an initial denaturation at 94°C for 2mins, followed by, 32 cycles of denaturation (94°C for 0.5min), annealing (64°C for 1min), extension (72°C for 1min), and a final elongation step (72°C for 2mins). Amplification products were resolved by agarose (2%) gel homeothermic electrophoresis at 80V for 30min. The predicted sizes of the amplification products were observed after electrophoresis, under an ultraviolet lamp, and densitometry was done. Then, the index of mRNA (RI) was calculated (RI=VEGF mRNA density/β-actin density x 100%).Detection of vascular endothelial growth factor positive neurons in dorsal root gangliaThe DRGs of each group were taken at 0, 3, 6, 12, 24, 48, 72, 96 hours and day seven after crushing the sciatic nerves. They were treated as follows (pv-6001/6002 immunohistochemistry kit from Beijing Zhongsan Jinqiao Biotechnology Ltd. China): Immunohistochemistry (1) APES (3-aminopropyl triethoxysilane) smear was conducted on glass slides; (2) Routine deparaffinizing was performed on the sections; (3) The sections were incubated in 3% H 2 O 2 in deionized water for 5-10mins to block endogenous peroxidase; (4) Hot-fix of the antigens was performed; (5) The primary antibody (Immediate-use VEGF rat anti-human monoclonal antibody from the immediate-use non-biotin pv-6001/6002 reagent kit, Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. Beijing, China) was added. The sections were placed in 4°C refrigerator overnight and then rinsed with phosphate buffered saline (PBS) three times for 2mins each; (6) The secondary antibody (Immediate-use goat antimouse IgG-horse radish peroxidase (HRP) polymers from the immediate-use nonbiotin pv-6001/6002 reagent kit, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was added. The sections were incubated at room temperature for 20-30mins, and then rinsed with PBS three times for 2mins each; (7) Diaminobenzidine (DAB) color development was conducted; (8) Washing with tap water was performed; (9) The samples were stained with hematoxylin, dehydrated, cleared and mounted. The sections were then observed under a microscope (50i, Nikon, Shanghai China) and the cells containing brown-yellow granules were considered to be positive. Image analysis Two specimens were randomly selected from each group at each time point and VEGF immunohistochemistry was performed. A light microscope (x400) was used to observe the number of cells with positive expression in each sample (five fields were randomly taken from each section to count the positive cells and the average value was calculated), and a comparison was conducted.Pathological examination The rats in each group were sacrificed at week four after crush. The DRGs and sciatic nerves were separated according to the method described. Examination of the DRGs and sciatic nerves was performed by electromicroscope.Nerve conduction velocities of sciatic nervesEight rats from each group were killed at week four after crush. Then a 6-cm length of sciatic nerve trunk was separated. The Nerve conduction velocities (NCVs) of sciatic nerves in the NCV determination box (constant temperature 35C) were detected at 2mins, 3mins, and 4mins, respectively (D95Super Lab determinator of NCV from Medical Electrons Institute of Academe of Jiangsu Biomedical Engineering). Different parameters of a stimulus impulse were: 0.04ms, 2.7voltage, and scanner speed 50,000mm/s. The distance between the two recording electrodes on every sciatic nerve trunk was 3.5cm. The time of action potential peak of two recording electrodes was detected.Toe spacesThe toe spaces of rats in each group were determined at week four after crush. We stained ink onto the rats' toes and made them walk on papers of 60cm length and 10cm width. The space of three to four toes of the right hind limbs was determined with vernier calipers.StatisticsStatistical Package for the Social Sciences (SPSS, Bizinsight, Beijing China) 11.5 was used, and Pvalues < 0.05 were considered to be significant. Analysis of variance (ANOVA) was used for comparison. The number of spinal cord neurons and the number of cells expressing VEGF were consistent with normal distributions. The Student-Newman-Keuls analysis (SNK) method was used.ResultsVascular endothelial growth factor mRNA by RT-PCRThere was little expression of VEGF in all groups, 0h after crushing, and no significant difference among them ( P >0.05). Compared with 0h: (1) the VEGF mRNA levels were not different from the control group ( P >0.05); (2) VEGF mRNA from other groups increased at 3h and 6h ( P < 0.01 at 6h) after crushing, and the VEGF mRNA from the saline, saline + VEGF antibody, alprostadil and alprostadil + VEGF antibody groups at 6, 12, 24, 48, 72, and 96 hours were more than that at 0h ( P < 0.01); (3) VEGF mRNA peaked at 72h and then decreased to baseline, such that there was no difference between 0h and day seven in each group ( P > 0.05); (4) VEGF mRNA from the saline, saline + VEGF antibody, alprostadil and alprostadil + VEGF antibody groups were significantly more than that of the control group at 6, 12, 24, 48, 72, and 96hours ( P < 0.01); (5) VEGF mRNA from the alprostadil and alprostadil + VEGF antibody groups were significantly more than that of the saline group at 6, 12, 24, 48, 72, and 96 hours ( P < 0.05), but there was no intragroup difference ( P > 0.05). There was no difference between the saline1VEGF antibody group and the saline group ( P > 0.05) [Figure 1],[Table 1].VEGF-positive neurons in DRGs There were only a few VEGF-positive neurons in the DRGs in the control group. In the alprostadil group, the number of VEGF-positive neurons began to increase at 6h ( P < 0.05), and at 12h in the other groups ( P < 0.01); the numbers peaked at 72h ( P < 0.01) and then started to decrease, returning to normal on day seven. The VEGF-positive neurons in the alprostadil group were more than that of the saline group at the same time ( P < 0.05). The VEGF-positive neurons in saline1VEGF antibody group were less than that of the saline group ( P < 0.05). The VEGF-positive neurons in the alprostadil + VEGF antibody group were less than that of the alprostadil group at the same time ( P < 0.05) [Figure 2],[Table 2].Pathological changes of DRGs electromicroscopicallyIn the saline and saline + VEGF-antibody groups, the density of electrons of numerous nuclei decreased, vacuolar degeneration and pyknosis appeared, the nucleoli or even nuclear structure disappeared, and the nuclear membrane was incomplete. The density of cytoplasm increased. The change of patching homogeneity, vacuolar degeneration, and many apoptosis body appeared in the cytoplasm. The pathological changes of the saline + VEGF-antibody group were more severe than those of saline group. In the alprostadil group, the density of electrons of some nuclei decreased and few apoptosis bodies appeared in the cytoplasm, and the other pathological changes were also less than those in the saline group. The pathological changes in alprostadil + VEGF-antibody group were severe than those in the alprostadil group. There was no pathological change of DRGs in the control group [Figure 3].NCVs of sciatic nervesThe NCVs of sciatic nerves in the alprostadil group were faster than that in the saline group ( P < 0.05) and slower than that in the control group ( P < 0.01) at week four after crush injury. The NCVs of sciatic nerves in the saline1VEGF-antibody group were slower than that in the saline group ( P < 0.05) and the NCVs of sciatic nerves in the alprostadil + VEGF-antibody group was slower than that in the alprostadil group ( P < 0.05) at week four after crush injuries [Table 3].Toe spacesAt week four after crush, the toe space in the saline group was significantly shorter than those in the control and alprostadil groups ( P < 0.01) and the toe space in the alprostadil group was not different to that in the control group ( P > 0.05). The toe space of the saline + VEGF-antibody group was shorter than that of the saline group, but there was no statistical value ( P > 0.05). The toe space of the alprostadil + VEGF-antibody group was shorter than that of the alprostadil group ( P <0.01) [Table 4].DiscussionRelevant research indicated that the expression of VEGF could be upregulated by anoxia. Following traumatic brain injury, there was an early (within 4h postinjury) increase in the expression of VEGF that was associated with neutrophilic invasion in the traumatized parenchyma. The maximum number of astrocytes expressing VEGF was observed on the fourth day after traumatic brain injury. [9] Macrophages in the periphery and the core of infarct represented the major source of VEGF during the early stages, 18h to day 2 postinjury. The number of immunoreactive macrophages decreased gradually from day 2, in both the periphery and core. VEGF was detected in neurons from 18h after the lesion to day five in the core, and up to day ten in the periphery. In the early stages, intracytoplasmic granular immunolabeling in the neurons and dendrites was observed. Immunostaining in glial cells was observed during the entire active period, from 18h after the lesion to day 14. However, their peak activities were occurred from day five to seven in the periphery. [10] Minamino reported that the expression of VEGF and other growth factors could be induced by hypoxia. [11] Gaumann found that the presence of VEGF was associated with tumor necrosis. Local hypoxia of tumors could induce VEGF and result in higher VEGFR-1 and VEGFR-2 levels in vascular endothelial cells. [12] Hypoxia increased the expression of VEGF mRNA and protein via the following mechanisms: (1) the regional promoter of VEGF included a response element of the hypoxia-inducible factor-1 (HIF-1). Hypoxia directly induced the expression of VEGF; (2) Stimulation of hypoxia activated c-Src, which increased the expression of VEGF; (3) During hypoxic induction, stability of VEGF was enhanced, and its half-life increased from 30-40min to 6-8h. This indicated that the increase in hypoxic-induced VEGF occurred on the transcriptional and post-transcriptional levels; (4) Mutations in the p53gene cause the survival of hypoxic tumors by increasing the expression of VEGF and decreasing the expression of thromboxane, and facilitating angiogenesis.[13] Vasoactive agents were commonly used in the clinic to treat crush injury to peripheral nerves, but the mechanism involved was unclear, until now. The results of this study demonstrated the vasoactive agent, alprostadil, could increase VEGF mRNA and VEGF-positive neurons in DRGs of rats after crush injury to sciatic nerves ( P <0.05). Despite that VEGF mRNA expression was not affected by the VEGF antibody results in our study, the VEGF-positive neurons in the saline + VEGF antibody group was less than that of the saline group ( P < 0.05), and the VEGF-positive neurons in alprostadil + VEGF antibody group was less than that in the alprostadil group ( P < 0.01). This indicated that the number of the VEGF- plus VEGF receptor-positive DRG neurons increased with alprostadil treatment, but decreased when the VEGF antibody was used. This might be because the biological activity of VEGF was inactivated or decreased when combined with the VEGF antibody. Altogether this suggested that alprostadil could upregulate VEGF and increase the combined expression of VEGF and VEGF receptors in DRGs after crush injury to sciatic nerves. Whether the expression of VEGF alone or in combination with VEGF receptors in DRG neurons was upregulated in the alprostadil group, needs to be further confirmed. It has been shown that VEGF was a key mediator of the angiogenesis in ischemic lesions in nervous tissues.[14],[15],[16] Quattrini observed that VEGF-A staining was significantly reduced in diabetic patients, compared with control subjects in the upper dermis and on blood vessels, and was the lowest in cases of severe neuropathy. VEGFR-2 staining did not differ in the dermis, but was significantly reduced on blood vessels of diabetic patients with severe neuropathy, compared with control subjects and diabetic patients without neuropathy.[17] Zheng reported that systemic administration of recombinant VEGF significantly diminished astrogliosis and increased the number of neuromuscular junctions in a Cu/Zn superoxide dismutase (SOD1) transgenic mouse model of amyotrophic lateral sclerosis treated with saline. [18] Uesaka demonstrated that angiogenesis is essential for the enlargement of any solid tumor, and VEGF is considered to be a major regulator. [19] Aramoto found that VEGF could induce mild vasodilation and obvious increases in microvascular permeability. [20] Cooke reported that VEGF could stimulate the release of nitric oxide from cultured human umbilical venous endothelial cells and upregulate the expression of nitric oxide synthase. [21] Recent evidence suggested that VEGF was protective against the effects of lesions in neurons or nerves in brain ischemia, [14],[22],[23],[24] spinal cord disorders [25] and even impairment of peripheral nerves. [14],[25],[26] If the blood flow of neurotrophic vessels of nerve was reduced by crush injury to nerve, oedema appeared in nerve tissues due to oxygen deficiency, the volume of nerve tissues increased, the pressure in neurolemma increased, the microcirculatory disturbance was further aggravated, the crush of nerves was aggravated, and the dysfunction of neural metabolism and conduction occurred. [12] Li [27] found that when neurolemma was dissected during facial nerve decompression, the oedematous nerves would immediately bulge, the blood vessels of the epineurium became dilate, the color of the nerves turned from pale to red, and facial movement would recover dramatically. The results of pathology, NCVs and toe spaces in this experiment indicated that alprostadil could reduce the pathological injury and improve the functional recovery after crushing injury to sciatic nerves.This study indicated that when the expressions of VEGF and the number of the VEGF- plus VEGF receptor-positive DRG neurons increased with alprostadil treatment, the pathological lesion of DRGs was alleviated and the functional rehabilitation of sciatic nerves was accelerated. In addition, the benefit of alprostadil was decreased by application of VEGF-antibody. It was suggested that the overexpressions of VEGF was important in pathological and functional repair of crush injury to sciatic nerves by alprostadil treatment.ConclusionsThe results of this experiment indicate that the vasoactive agent alprostadil may reduce the pathological lesion of peripheral nerves and improve the rehabilitation of the neural function, which may relate to upregulation of the expression of VEGF, following crush injury to peripheral nerves.AcknowledgmentThis work was supported by the Medical Research Council [Grant number: Natural Science of Jiangsu Province BK2001116]. References1.Ryderik B, Lundborg G. Permeability of intraneural microvessels and perineurium following acute graded experimental nerve compression. Scand J Plast Reconstr Surg 1977;11:179-85. 2.Bischoff B, Romstck J, Fahlbusch R, Buchfelder M, Strauss C. Intraoperative brainstem auditory evoked potential pattern and perioperative vasoactive treatment for hearing preservation in vestibular schwannoma surgery. J Neurol Neurosurg Psychiatry 2008;79:170-5. 3.Uehara K, Sugimoto K, Wada R, Yoshikawa T, Marukawa K, Yasuda Y, et al. Effects of cilostazol on the peripheral nerve function and structure in STZ-induced diabetic rats. J Diabetes Compl 1997;11:194-202. 4.Kihara M, Low PA. Vasoreactivity to prostaglandins of rat peripheral nerve. J Physiol 1995;484:463-7. 5.Shirasaka M, Takayama B, Sekiguchi M, Konno S, Kikuchi S. Vasodilative effects of prostaglandin E1 derivate on arteries of nerve roots in a canine model of a chronically compressed cauda equina. BMC Musculoskelet Disord 2008;9:41. 6.Milio G, Cospite V, Cospite M. Effects of PGE-1 in patients suffering from peripheral arterial occlusive disease. Minerva Cardioangiol 2003;51:311-6. 7.Umemura K, Nakashima M. Effect of prostaglandin E1 on the rat inner ear microvascular thrombosis. Gen Pharmacol 1997;28:221-4. 8.Patro IK, Hattopadhyay M, Patrio N. Flunarizine enhances functional recovery following sciatic nerve crush lesion in rats. J Neurosci Lett 1999;26:97-100. 9.Chodobski A, Chung I, Kozniewska E, Ivanenko T, Chang W, Harrington JF, et al. Early neurotrophilic expression of VEGF after brain traumatic injury. Neuroscience 2003;122:853-67. 10.Kovacs Z, Ikezaki K, Samoto K, Inamura T, Fukui M. VEGF and flt Expression time kinetics in rat brain infarct. Stroke 1996;27:1865-73. 11.Minamino T, Tateno K. Theraputic angiogenesis for critical limb ischemia by implantation of peripheral mononuclear cells. Med Devel (Jap) 2006;217:397-401. 12.Gaumann AK, Schermutzki G, Mentzel T, Kirkpatrick CJ, Kriegsmann JB, Konerding MA. Microvessel density and VEGF/VEGF receptor status and their role in sarcomas of the pulmonary artery. Oncol Rep 2008;19:309-18. 13.Wen HM, Huang RX. Specific vascular growth factor and treatment of ischemic cerebrovascular disease. Abroad Mede Fascicle Cerebrovasc Dis 2004;12:204-7. 14.Zachary I. Neuroprotective role of vascular endothelium growth factor: Signalling mechanisms, biological function, and therapeutic potential. Neurosignals 2005;14:207-21. 15.Wang Y, Kilic E, Kilic U, Weber B, Bassetti CL, Marti HH, et al. VEGF overexpression induces post-ischemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain 2005;128:52-63. 16.Shintani S, Murohara T. Theraputic angiogenesis by cell transplatation. Med Devel (Jap) 2006;217:392-6. 17.Quattrini C, Jeziorska M, Boulton AJ, Malik RA. Reduced vascular endothelial growth factor expression and intra-epidermal nerve fiber loss in human diabetic neuropathy. Diabetes Care 2008;31:140-5. 18.Zheng C, Skald MK, Li J, Nennesmo I, Fadeel B, Henter JI. VEGF reduces astrogliosis and preserves neuromuscular junctions in amyotrophic lateral sclerosis transgenic mice. Biochem Biophys Res Commun 2007;363:989-93. 19.Uesaka T, Shono T, Suzuki SO, Nakamizo A, Niiro H, Mizoguchi M, et al. Expression of VEGF and its receptor genes in intracranial schwannomas. J Neurooncol 2007;83:259-66. 20.Aramoto H, Breslin JW, Pappas PJ, Hobson RW 2 nd , Durán WN. Vascular endothelium growth factor stimulates differential signalling pathways in in vivo microvascular. Am J Physiol Heart Circ Physiol 2004;287:H1590-8. 21.Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;105:2133-5. 22.Sun FY, Guo X. Molecular and cellular mechanisms of neuroprotection by vascular endothelium growth factor. J Neurosci Res 2005;79:180-4. 23.Storkebaum E, Lambrechts D, Carmeliet P. VEGF once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 2004;26:943-54. 24.Kilic E, Kilic U, Wang Y, Bassetti CL, Marti HH, Hermann DM. The phosphatidylinositol-3 kinase/Akt pathway VEGFs neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia. FASEB J 2006;20:1185-7. 25.Ding XM, Mao BY, Jiang S, Li SF, Deng YL. Neuroprotective effect of exogenous vascular endothelium growth factor on rat spinal cord neurons in vitro hypoxia. Chin Med J (Engl) 2005;118:1644-50. 26.Hasegawa T, Kosaki A, Shimizu K, Matsubara H, Mori Y, Masaki H, et al. Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats. Exp Neurol 2006;199:274-80. 27.Li JD, Li XP. To survey microcirculation of facial nerve of rabbit by laser-Doppler flow imaging. J China Otolaryngol 2002;37:184-7. Figures Tables
2016-08-22小哥江苏健康广播世界卫生组织调查结果显示中国脑卒中(俗称中风,包括脑梗死、脑出血、蛛网膜下腔出血等)发病率排名世界第一,我国国内调查结果亦表明脑卒中为中国国民第一位死因。脑卒中除了高发病率和高致死率外,还具有高致残率和高复发率的特点,严重威胁着国民的生命和健康。其中缺血性卒中,亦即脑梗死(脑梗塞)最多见,约占80%。近年来脑卒中的发病有年轻化趋势,大家应高度警惕!今天,我们就缺血性卒中的病因、急救和预防跟大家作一介绍。缺血性卒中的病因与危险因素有哪些?已知的缺血性卒中的病因:最常见的是动脉粥样硬化;其次是高血压病伴发的脑小动脉硬化;其他的如动脉壁的炎症等。缺血性卒中的危险因素包括:不可控制的危险因素,如年龄、性别、家族倾向、种族和遗传;和可控制的危险因素,常见的如高血压病、高同型半胱氨酸、糖尿病、心脏疾病、高脂血症和肥胖、吸烟和酗酒、血液成分异常(如真性红细胞增多症、血小板增多等)。另,对某些特殊人属如育龄期妇女,口服避孕药物,应知其可诱发卒中。缺血性卒中如何进行急诊救治?抢救缺血性卒中必须争分夺秒地进行,最好能在3小时内或更短时间内确诊并得到有效治疗。这可以极大地减少后遗症的发生。如此,大家必须做到“五早”,即早就诊、早诊断、早治疗、早康复、早预防,其中早就诊与早预防是广大市民应予重视的。早就诊 就是要重视脑卒中、提高警惕性。许多病人出现头晕、言语不清、肢体活动不灵活、麻木、血压突然升高等症状,心存侥幸,不想到医院而等待自行缓解,结果症状越来越重,延误了最佳抢救时间。因此,及时发现、迅速送诊是争取早期治疗的关键。这里要指出的是在送诊时,建议大家尽可能:第一抢时间;第二选择120来转运病人,而不要选择自我送诊。早预防 患病总不如不发病,没有病防止发病称一级预防,已患过病防止再发为二级预防。缺血性卒中的急救关键是血运重建及其在此基础上的神经保护,其治疗手段可分为药物干预和非药物治疗(包括外科手术),两者又是相辅相成的。如下为目前指南推荐的缺血性卒中常用的关键性的急诊救治措施:超早期/早期的溶栓/机械取栓:这是最积极的血管再通治疗策略。近年指南已将溶栓的时间窗放宽为起病后3-4.5小时,又分为动脉(介入技术介导下的)溶栓和静脉溶栓。但溶栓的时间窗很窄,且有很多的禁忌症,同时还必须得到家属的知情同意,因此,能够接受溶栓治疗的病人很少。近年来,介入机械取栓这项措施悄然兴起,因其时间窗相对较宽,可使更多患者获益。这里要指出的是介入治疗业已被广泛应用于临床对缺血性卒中的诊断和治疗,推动了缺血性卒中的临床诊疗进展。抑制血小板聚集:这是目前临床上治疗缺血性卒中最普遍的再通策略之一,抑制血小板聚集的药物有阿司匹林、氯吡格雷、双嘧达莫等。开通/改善侧枝循环/微循环:这是目前临床上治疗缺血性卒中最普遍的再通策略之一,不少血管活性药物(包括部分提纯至单体的中草药制剂)有此功效,血压的管理应是其中重要的一环。神经保护治疗:神经保护剂很多,基础研究的结果都是肯定的,在临床实践中可见许多患者获益,但尚未见统一的意见,仍在探索之中。祖国传统医药:中医中药被广泛应用于缺血性卒中的治疗已多年,不足的是多中心双盲对照研究的循证医学研究较少。缺血性卒中应如何预防?脑卒中经过急性期救治病情稳定后即进入恢复期。此刻即应在专科大夫的指导下进行规范的卒中二级预防。对于缺血性脑卒中,分为一级预防和二级预防。一级预防的目的是“防患于未然”,就是寻找、去除脑卒中的病因与危险因素,而二级预防目的则已是“消弥于已然”,可以用两句话来概括:其一,寻找、去除如上所述的缺血性卒中的病因与危险因素;其二,对暂短性脑缺血发作以及已经缺血性卒中的患者,除了规范应用抗拴药物(如抑制血小板聚集制剂、抗凝药物、中药等)外,针对动脉粥样硬化所致的血管狭窄不仅可以应用他汀类药物,还可以采用颈动脉内膜切除术或者血管内支架成形术来预防卒中。另,特殊人群应做相应预防措施。专访专家:唐金荣,男,医学博士,主任医师,研究生导师。中国卒中学会会员,江苏省医学会脑血管病学组委员,Neuroscience Letters 审稿人及中华临床医师杂志特约审稿专家。从事神经内科临床、教学及科研工作近30年,对神经内科各种常见疾病和危重疑难疾病的诊断和治疗积累了丰富的临床经验,尤其在脑血管病、周围神经疾病、中枢神经系统感染以及头痛等疾病的诊断和治疗方面有较深的研究,并指导研究生的学习。负责完成江苏省重大招标项目(BS2006007)卒中急救绿色通道的建设、卒中急救规范以及卒中急救关键技术方案的制定和实施;主持完成一项江苏省自然基金资助项目(BK2001116);在SCI收录期刊及神经病学专业中文核心期刊发表论文70余篇;近年来获卫生厅新技术引进奖一等奖及市级科技进步奖数项。专家门诊时间:每周二、周三、周六上午
卒中(Stroke)又称中风,是危害全人类身心健康和生命安全的三大主要疾病之一,其发病率、致死率、致残率和复发率之高也已成为全球关注的焦点之一,而且其合并症、并发症也多。目前,卒中救治的措施是层出不穷、理论也在日渐更新。本文介绍的是有组织地合理运用卒中救治措施的组织或系统——卒中中心(stroke center)。2000年美国脑发作联盟(brain attack coalition,BAC)讨论了卒中中心(stroke center,SC)的概念,并推荐了2种类型:初级卒中中心(primary stroke center, PSC)和综合卒中中心(comprehensive stroke center,CSC)。PSC包括必要的人员、机构和绝大多数急诊卒中患者诊治的流程。虽然PSC能够为卒中患者提供高质量的医疗,但是典型的PSC中无法为疑难的卒中类型、严重功能缺失或多器官损害患者提供必要的特殊医疗及技术支持。这些患者往往需要经过特殊训练的内科医生和各专业的专家采取先进的诊断和治疗手段。CSC是一个组织或系统,需要专门的人员队伍、专家、基础设施以及卒中诊治程序,例如高质量的内、外科医疗、特殊检查或介入治疗。不仅大面积缺血性卒中和出血性卒中患者,而且病因少见或者是需要特殊治疗或多科室联合治疗的卒中患者也能会CSC中受益。CSC的附加功能是作为该地区其他医疗组织,如PSC的资源中心,这包括提供治疗特殊病例的专家、指导患者分诊、指导PSC的检查与治疗、为该城市(地区)其他医院和卫生专业人员提供教育资源。为发展、完善综合卒中中心/系统,BAC正积极对各级医院、卫生专业人员和管理人员进行指导。CSC有其必须的的要素、要求,以及执行规程。各个组织(机构)和健康支持系统可在此基础上发展自己的CSC,努力形成与当地实际相符合的CSC。在医疗质量方面,PSC与CSC应没有区别,每个人(包括各级医院、卫生专业人员、健康支持系统及患者)对这两个中心的预期值都很高。(原文登载在《中国卒中杂志》2007年 第2卷 第11期 925-937页)
在卒中的预防中,血压下降与终点事件之间存在正性相关,但并非越低越好,越来越多的循证证据支持降压治疗中存在“J”型曲线。所谓“J”型曲线现象,即血压下降达到特定水平时,主要心、脑血管疾病的发生率会下降;但继续再降低血压,心脑血管事件发生率反而会回升。当血压在130-139/80-85 mmHg 左右时,心脑血管事件的发生率较低,这为确定理想的降压目标提供了证据。为了降压达标,有时需要联合几种降压药治疗。