Sarin-induced brain damage in rats is attenuated by delayed administration of midazolam

Sarin poisoned rats display a hyper-cholinergic activity including hypersalivation, tremors, seizures and death. Here we studied the time and dose effects of midazolam treatment following nerve agent exposure. Rats were exposed to sarin (1.2 LD50, 108 μg/kg, im), and treated 1 min later with TMB4 and atropine (TA 7.5 and 5 mg/kg, im, respectively). Midazolam was injected either at 1 min (1 mg/kg, im), or 1 h later (1 or 5 mg/kg i.m.). Cortical seizures were monitored by electrocorticogram (ECoG). At 5 weeks, rats were assessed in a water maze task, and then their brains were extracted for biochemical analysis and histological evaluation. Results revealed a time and dose dependent effects of midazolam treatment. Rats treated with TA only displayed acute signs of sarin intoxication, 29% died within 24 h and the ECoG showed seizures for several hours. Animals that received midazolam within 1 min survived with only minor clinical signs but with no biochemical, behavioral, or histological sequel. Animals that lived to receive midazolam at 1 h (87%) survived and the effects of the delayed administration were dose dependent. Midazolam 5 mg/kg significantly counteracted the acute signs of intoxication and the impaired behavioral performance, attenuated some of the inflammatory response with no effect on morphological damage. Midazolam 1 mg/kg showed only a slight tendency to modulate the cognitive function. In addition, the delayed administration of both midazolam doses significantly attenuated ECoG compared to TA treatment only. These results suggest that following prolonged seizure, high dose midazolam is beneficial in counteracting adverse effects of sarin poisoning.     

Choice of approaches in developing novel medical countermeasures for nerve agent poisoning

During the establishment of a research branch, all relevant matters encountered will be of interest to study. After having acquired a body of basal knowledge, it becomes possible to derive ideas or hypotheses for further elaboration of information. The purpose of the present study was to show that therapies for nerve agent poisoning based on specific neuropharmacological approaches can have greater probability for being successful than treatment regimens based on fragmental research or serendipitous discoveries. By following the guidelines for research in experimental epilepsy, neuronal target areas for nerve agents have been identified through lesion studies, and critical receptors for pharmacological treatment have been specified through microinfusion studies of rats. Subsequent experimentations have shown that the results achieved from microinfusion studies are transferable to systemic administration. It is demonstrated that a treatment regimen developed through the novel approach is more efficacious than regimens derived from conventional research on countermeasures. A therapy consisting of HI-6, levetiracetam, and procyclidine that has been worked out along the new lines, exerts powerful anticonvulsant capacity and appears to have universal utility as a stand-alone therapy against soman intoxication in rats. It would be of great interest to examine whether the latter findings can be expanded to other animal species than rats and other classical nerve agents than soman.     

Primary brain targets of nerve agents: The role of the amygdala in comparison to the hippocampus

Exposure to nerve agents and other organophosphorus acetylcholinesterases used in industry and agriculture can cause death, or brain damage, producing long-term cognitive and behavioral deficits. Brain damage is primarily caused by the intense seizure activity induced by these agents. Identifying the brain regions that respond most intensely to nerve agents, in terms of generating and spreading seizure activity, along with knowledge of the physiology and biochemistry of these regions, can facilitate the development of pharmacological treatments that will effectively control seizures even if administered when seizures are well underway. Here, we contrast the pathological (neuronal damage) and pathophysiological (neuronal activity) findings of responses to nerve agents in the amygdala and the hippocampus, the two brain structures that play a central role in the generation and spread of seizures. The evidence so far suggests that exposure to nerve agents causes significantly more damage in the amygdala than in the hippocampus. Furthermore, in in vitro brain slices, the amygdala generates prolonged, seizure-like neuronal discharges in response to the nerve agent soman, at a time when the hippocampus generates only interictal-like activity. In vivo experiments are now required to confirm the primary role that the amygdala seems to play in nerve agent-induced seizure generation.     

Apoptosis of spinal interneurons induced by sciatic nerve axotomy in the neonatal rat is counteracted by nerve growth factor and ciliary neurotrophic factor

We have previously shown that not only motoneurons and dorsal root ganglion cells but also small neurons, presumably interneurons in the spinal cord, may undergo apoptotic cell death as a result of neonatal peripheral nerve transection in the rat. With the aid of electron microscopy, we have here demonstrated that apoptosis in the spinal cord is confined to neurons and does not involve glial cells at the survival time studied (24 hours). To define the relative importance of the loss of a potential target (motoneuron) and a potential afferent input (dorsal root ganglion cell) for the induction of apoptosis in interneurons in this situation, we have compared the distributions and time courses for TUNEL labeling, which detects apoptotic cell nuclei, in the L5 segment of the spinal cord and the L5 dorsal root ganglion after sciatic nerve transection in the neonatal (P2) rat. In additional experiments, we studied the effects on TUNEL labeling of interneurons after treatment of the cut sciatic nerve with either ciliary neurotrophic factor (CNTF) to rescue motoneurons or nerve growth factor (NGF) to rescue dorsal root ganglion cells. The time courses of the TUNEL labeling in motoneurons and interneurons induced by the lesion show great similarities (peak at 8-48 hours postoperatively), whereas the labeling in dorsal root ganglion cells occurs later (24-72 hours). Both CNTF and NGF decrease the number of TUNEL-labeled interneurons, but there is a regional difference, in that CNTF preferentially saves interneurons in deep dorsal and ventral parts of the spinal cord, whereas the rescuing effects of NGF are seen mainly in the superficial dorsal horn. The results are interpreted as signs of a trophic dependence on both the target and the afferent input for the survival of interneurons neonatally.     

神经生长因子对糖尿病神经病变大鼠神经肽和神经传导速度的影响

目的 探讨神经生长因子对糖尿病周围神经病变大鼠神经肽和神经传导速度的影响. 方法 雄性Wistar大鼠35只按随机数字表法分为健康对照组(n=10)、糖尿病模型组(n=13)和神经生长因子治疗组(n=12),后两组用链脲佐菌素制成糖尿病周围神经病变大鼠模型,并给予神经生长因子治疗组神经生长因子治疗(40μg/kg).显微镜下观察并计算背根神经节中P物质、降钙素基因相关肽(CGRP)免疫阳性细胞率,检测运动神经传导速度(MNCV)和感觉神经传导速度(SNCV). 结果 糖尿病模型组大鼠背根神经节中P物质、CGRP免疫阳性细胞率(27.710％±3.471％;36.360％±12.027％)以及神经生长因子治疗组治疗前MNCV [(35.80±6.19) m/s]、SNCV[(39.62±6.69) m/s]与健康对照组[P物质:44.225％±8.213％;CGRP:47.400％±13.723％;MNCV:(55.83±10.30) m/s; SNCV:(47.02±7.52) m/s]相比显著下降,差异有统计学意义(P＜0.05).经神经生长因子治疗后,P物质、CGRP免疫阳性细胞率(49.417％±6.753％;53.811％±7.125％)较糖尿病模型组显著增高,MNCV[(41.80±3.45) m/s]、SNCV[(42.92±6.69) m/s]均治疗前显著增高,差异有统计学意义(P＜0.05). 结论 糖尿病周围神经病变大鼠可出现神经传导速度下降和神经生长因子相关神经肽P物质、CGRP缺乏,而神经生长因子可促进神经肽的表达并提高神经传导速度.     

神经桥接与导管套接修复猫动眼神经形态学观察

目的探求导管修复颅内段动眼神经的可行性.方法 20只健康家猫随机分为2组.将右侧动眼神经于脑池段切断后,分组采用自体神经桥接和导管套接的方法修复.术后14周末光镜、电镜观察神经纤维的连续性、再生纤维数目和直径.结果神经修复14周后,神经桥接组67%、导管套接组75%的动物其动眼神经功能均有一定程度的恢复.形态学显示两组均取得较好的神经再生效果,两组间再生纤维直径差异无显著性(P>0.05),但导管套接组再生神经纤维数目较多,差异有显著性(P<0.05).结论导管套接法可作为颅内段动眼神经损伤后的一种修复方法,其疗效近似或稍优于神经桥接法.     

Weak electric current accelerates motoneuron regeneration in the sciatic nerve of ten-month-old rats

When a weak direct current (DC) of 10 μA driven by an implantable device was applied to a crushed sciatic nerve in 9–10-month-old rats, regeneration was accelerated as assessed electrophysiologically. Rats underwent a sciatic nerve crush of the right leg and were randomly assigned to one of three groups: (i) Cathode Distal (CD); (ii) Sham Implant (SI); or (iii) No Implant (NI). On post-operative days 25–39 each rat r received a measurement of evoked electromyograms (EMG) in the foot. In addition each rat received a behavioural test score (BTS). This BTS correlated well with the evoked EMG results showing the behavioural relevance of the EMG results. NI rats performed the same as SI in the evoked EMG procedure, but CD rats performed significantly better than both the control groups. The recovery period in the CD rats was 21% shorter than in either the sham-treated or untreated controls showing that weak DC can greatly enhance nerve regeneration in adult rats. This result was in contrast to previous work in younger rats where DC treatments had no effects. The discrepancy is discussed in relation to the slowing of regeneration in older rats.     

Maturation of the spontaneous transmitter release by regenerated nerve endings in vitamin E-deficient rats

Summary In order to verify the importance of the protection against lipid peroxidation in presynaptic differentiation and maturation, the reappearance and maturation of the spontaneous transmitter release during the extensor digitorum longus muscle reinnervation following a lesion of the sciatic nerve were studied in normal and vitamin E-deficient rats. The study was carried out by intracellular recordings in order to observe the miniature end plate potentials in the reinnervated end plates.In control and vitamin E-deficient rats the first signs of muscle innervation reappeared simultaneously, but in the latter the spontaneous transmitter release mechanism matured more slowly; furthermore, in the long-term, very low mepp frequencies continued to occur. The data suggest a slowing of the transmitter release mechanism maturation and a protracted rearrangement of innervation in the deficient rats.     

Suppression of experimental allergic neuritis in rats by prior immunization with nerve in saline

Summary Rats given nerve in saline intraperitoneally failed to develop allergic neuritis when challenged with nerve in adjuvant 2 weeks later. The serum of protected rats prevented sensitized lymph node cells, from rats immunized only with nerve in adjuvant, from demyelinating rat peripheral nerve cultures.This work was supported in part by Special Fellowship NB 01787 from the National Institute of Neurological Diseases and Stroke (Dr. Lehrich), and by USPHS Grant NDS 06021 and Grant 428 from the National Multiple Sclerosis Society.     

Regulation of myelinated nociceptor function by nerve growth factor in neonatal and adult rats

The role of nerve growth factor (NGF) as a survival factor for sensory neurons during embryonic life has been well documented. Here we examine the actions of NGF or antisera against NGF (anti-NGF) on physiologically identified sensory neurons with myelinated axons later in life, after the dependence on NGF for survival ends. We find that the effects of NGF and anti-NGF are specific for sensory neurons which are nociceptors. Treatments were found to affect the biophysical properties, the development, or the physiological function of myelinated nociceptors. They also affect the animal's behavioral response to noxious stimulation, depending upon when the treatments were given: neonatally, from 2–5 weeks of age, or chronically, beginning at birth. Thus, we find that the actions of NGF are specific for nociceptors but that the function of this neurotrophic factor changes according to the developmental age of the animal.     

Modification of the peripheral nerve disturbance in ciguatera poisoning in rats with lidocaine

Electrophysiological studies were performed on the ventral tail nerve of adult rats following intraperitoneal injection of a crude extract of ciguatoxin from known toxic fish flesh. Ciguatoxin induced significant slowing of both mixed and motor nerve conduction velocities and also significant reductions in both motor and mixed nerve amplitudes. Both absolute and supernormal periods were significantly prolonged together with an increase in the magnitude of the supernormal response. These electrophysiological disturbances were modified or blocked by intraperitoneal lidocaine. These findings suggest that lidocaine may have a potential therapeutic application in the treatment of the neurological disturbance in acute ciguatera poisoning in humans.     

Collateral reinnervation of skin by C-fibres following nerve injury in the rat

Evidence for collateral reinnervation of skin by saphenous nerve C-fibres, following either crush or section of the ipsilateral sciatic nerve, was obtained in the rat using a dye-labelled plasma extravasation technique. During the period of regeneration of the crushed nerve the spread of collateral reinnervation was halted and may even have been reversed.     

神经减压缓解糖尿病大鼠触诱发痛的机制研究

目的探讨周围神经减压术缓解糖尿病大鼠触诱发痛的机制。方法将健康成年雄性SD大鼠按随机数字表法分为5组,分别为Ⅰ组(正常对照,n=10)、Ⅱ组(糖尿病模型,n=20)、Ⅲ组(糖尿病模型+乳胶管置入,n=10)、Ⅳ组(糖尿病模型+乳胶管置入+神经减压,n=10)及Ⅴ组(糖尿病模型+乳胶管置入+单纯术区显露,n=10)。糖尿病模型采用链脲佐菌素(STZ)腹腔注射,神经减压法即去除坐骨神经乳胶管。建模后3 d,采用up-down法检测各组大鼠的缩足阈值,保留Ⅱ~Ⅴ组中出现触诱发痛的大鼠。采用透射电镜观察5组大鼠坐骨神经的形态,分别对有髓和无髓神经纤维进行测量。进一步采用蛋白质免疫印迹(WB)和免疫荧光实验对5组大鼠脊髓后角γ-氨基丁酸B型(GABAB)受体的表达进行定量和定位检测。结果术后3周,STZ注射结合乳胶管置入(Ⅲ、Ⅳ、Ⅴ组)较单纯性STZ注射(Ⅱ组)大鼠触诱发痛的发生率高[分别为86.7%(26/30)、55.0%(11/20),χ^2=6.254,P=0.012]。Ⅱ、Ⅲ、Ⅳ、Ⅴ组的缩足阈值[分别为(4.06±1.28)g、(3.09±1.43)g、(4.02±1.96)g、(4.15±1.87)g]均低于Ⅰ组[(13.41±1.88)g,均P<0.05];术后5周,Ⅳ组的缩足阈值高于Ⅱ、Ⅲ、Ⅴ组(均P<0.05)。电镜观察结果显示,Ⅱ、Ⅲ、Ⅳ、Ⅴ组有髓和无髓神经纤维的面积、密度均较Ⅰ组减小(均P<0.05),有髓神经纤维的g比例较Ⅰ组增加(均P<0.05);Ⅳ组有髓纤维的面积和密度均大于Ⅱ、Ⅴ组(均P<0.05)、g比例低于Ⅱ、Ⅴ组(均P<0.05)。WB结果显示,神经减压术后3周,Ⅱ、Ⅲ、Ⅳ、Ⅴ组GABAB受体的表达量均较Ⅰ组下降(均P<0.05),而Ⅳ组高于Ⅴ组(P<0.05)。免疫荧光结果显示,Ⅱ、Ⅲ、Ⅳ、Ⅴ组中GABAB受体在脊髓背角内神经丝蛋白(NF)-200+区和NF-200+神经元的表达均较Ⅰ组下调(均P<0.05)。结论周围神经减压术可缓解糖尿病大鼠触诱发痛,主要通过去除有髓神经纤维的压迫、解除GABAB受体下调介导的中枢敏化,从而恢复脊髓兴奋性升高的病理状态。     

Anticonvulsant efficacy of antihistamine cyproheptadine in rats exposed to the chemical warfare nerve agent soman

Organophosphate compounds, such as soman and sarin, are highly toxic chemical warfare nerve agents that cause a build-up of acetylcholine in synapses and neuromuscular junctions. Current therapies aim to prevent seizures and protect against brain injury following exposure. The present study was designed to evaluate the effectiveness of the antihistamine cyproheptadine in improving survival and controlling seizures in rats exposed to soman. Rats were pretreated with the oxime reactivator HI-6 (125 mg/kg, ip) 30 min prior to soman exposure (225 μg/kg, sc) and then treated with atropine methylnitrate (AMN, 2.0 mg/kg, im) 1 min after soman. Cyproheptadine (10, 13, 16 or 20 mg/kg, ip) was given at one of three time points: 1 min after soman intoxication, at the onset of soman-induced seizures or 5 min after seizure onset. Control animals were exposed to soman and given an equivalent volume of sterile water instead of cyproheptadine. The incidence of seizures, mortality, neuron counts, neuropathology and apoptosis in specific regions of the brain were evaluated. In animals given HI-6 and AMN the incidence of soman-induced seizure and mortality rate within the first 24 h were 100%. When cyproheptadine was given at a dose of 13 or 20 mg/kg 1 min after soman exposure, the incidence of seizures was reduced from 100% to 13% and 30%, respectively. In addition, cyproheptadine given at 1 min after soman exposure increased the survival rate to 100% regardless of dose. When cyproheptadine was administered at seizure onset, seizures were terminated in 100% of the animals at doses above 10 mg/kg. The survival rate with cyproheptadine treatment at the onset of seizure was ≥83%. Seizures terminated in ≥75% of the animals that received cyproheptadine 5 min after soman-induced seizure onset. When given at 5 min after seizure onset the survival rate was 100% at all tested doses of cyproheptadine. The neuropathology scores and the number of TUNEL positive cells in the brain regions examined decreased at all time points and cyproheptadine doses tested. These observations indicate that cyproheptadine treatment can effectively control seizures, improve survival, reduce seizure duration and reduce the number of dying cells in the brain following soman exposure.     

Chronic stimulation of the peroneal nerve in rats upregulates the pro-opiomelanocortin gene in spinal motoneurones

Continuous unilateral stimulation of the peroneal nerve in rats for 8 h per day for 2 or 7 days caused significant increases in POMC mRNA and beta-endorphin immunoreactivity in both ipsilateral and contralateral motoneurones. Intermittent stimulation, for 10-min periods with 90-min rest periods, for 8 h per day for 2 days also caused upregulation of POMC mRNA. It is suggested that expression of POMC-derived peptides in motoneurones may be important for maintaining muscle contractile function.     

不同剂量神经生长因子对神经源性疼痛大鼠脊髓Fos蛋白的影响

背景：神经生长因子对神经元的存活、生长发育、分化、再生和功能维持起到调控作用。 目的：进一步验证不同剂量神经生长因子对神经源性痛大鼠的治疗作用以及对脊髓中Fos蛋白的影响。 设计、地点：随机对照动物实验，在上海第九人民医院完成。 材料：健康成年雄性Wistar大鼠72只,体质量180~200 g，随机分为4组，模型组及腹腔注射神经生长因子4， 8，16 μg组，每组18只。 方法：72只大鼠结扎左侧坐骨神经制备坐骨神经慢性缩窄性损伤模型；腹腔注射神经生长因子4， 8，16 μg组，造模后分别腹腔注射神经生长因子4，8，16 μg/(kg•d)。 主要观察指标：①于术前和术后第1，2，3，5，7，10，14天进行行为学观察和机械性痛阈测定。②于术后第2，7，14天各组分别处死大鼠各6只，取脊髓，应用免疫组织化学方法和图像分析系统检测脊髓中Fos蛋白的表达。 结果：模型组大鼠术后出现自发抬足、舔足等自发痛表现，术后第3天起开始出现痛阈下降，第14天达最低值，而注射神经生长因子组大鼠没有自发痛表现，没有出现机械性痛阈的低常期，第10，14天模型组大鼠痛阈与其余各组比较差异有显著性意义(P < 0.05)。4组大鼠术后第1天机械性痛阈普遍升高；注射神经生长因子16 μg组大鼠术后第1天机械性痛阈比其他各组低(P < 0.01)，第2天机械性痛阈恢复至术前水平，低于注射神经生长因子4 μg组(P < 0.05)。术后模型组大鼠脊髓Fos蛋白表达进行性升高，而其他各组大鼠脊髓中仅有少量Fos阳性神经元，模型组与其余各组比较差异显著(P < 0.01)。术后第2天注射神经生长因子16 μg组大鼠脊髓Fos蛋白表达最低，与模型组和注射神经生长因子4 μg组比较，差异有显著性意义(P < 0.01)。 结论：神经生长因子对大鼠神经源性痛有治疗作用，且大剂量较小剂量作用更为明显，其机制可能与抑制脊髓中Fos蛋白表达有关。     

The effect of vincristine on nerve regeneration in the rat. An electrophysiological study

Weekly injections of vincristine to produce a dose-dependent delay in regeneration following sciatic nerve crush. With 20 micrograms/kg/wk recovery was similar to that in control animals. With 50 and 100 micrograms/kg/wk electrophysiological evidence of reinnervation of the foot muscles was significantly delayed and muscle action potential amplitude increased at a slower rate. However, once begun the increase in motor nerve conduction velocity was closer to that in control animals. With 200 micrograms/kg/wk no evidence of reinnervation of the foot muscles was found even after 6 months. These doses produced no abnormality of muscle action potential amplitude or of nerve conduction velocity on the opposite non-crushed side.     

Effect of nerve growth factor on the lesioned septohippocampal cholinergic system of aged rats

Nerve growth factor (NGF) was injected intraventricularly into aged (24 months) rats with unilateral lesions of the lateral fimbria. The activity of choline acetyltransferase (ChAT) was determined in the septum and hippocampus from the normal unlesioned rats, lesioned and cytochrome c-treated rats (controls), and lesioned and NGF-treated rats at different times after the lesion. NGF-injection for 15 days after the lesion resulted in an increase of the ChAT activity in both the contralateral hippocampus and the entire septum, to about 130% of that in the normal animals, but resulted in a slight increase in the ipsilateral lesioned hippocampus, when compared to the activity in the ipsilateral side of the cytochrome c-treated controls. NGF-injection for 30 days after the lesion resulted in a 48% increase of the ChAT activity in the ipsilateral hippocampus as compared to cytochrome c-treated controls, but failed to result in a significant increase in the contralateral hippocampus. These findings indicate that atrophic cholinergic neurons in aged animals are similarly responsive to NGF treatment, like these in the young animals. Moreover, these findings suggest that the responses of basal forebrain cholinergic neurons to NGF treatment varies with time after the lesion and imply that the NGF administration can promote the collateral sprouting from spared cholinergic fibers after the lesion in the aged forebrain.     

Thinning of cervical nerve roots and peripheral nerves in ALS as measured by sonography

ObjectiveProgressive atrophy and loss of motor axons is a hallmark of amyotrophic lateral sclerosis (ALS). Limited sonographic data are available on potential detection of atrophy of peripheral nerves and nerve roots in ALS.MethodsPatients with either definite or probable ALS and control subjects underwent sonographic evaluation of the cervical roots (C5, C6, and C7) and peripheral nerves (median and ulnar nerves) on the right. These diameters and cross-sectional areas (C6, median, and ulnar nerves) were compared.ResultsThe diameters and cross-sectional areas were consistently smaller in ALS than in controls. No correlation was present between the sonographic parameters and the disease severity, disease duration, age, or gender. The overall sensitivity and specificity tended to be greater in the cervical nerve roots than in the peripheral nerves.ConclusionsThis study shows atrophy of cervical nerve roots and peripheral nerves in ALS detected by sonography. Cervical nerve roots might be more appropriate to detect motor axon loss than peripheral nerves.SignificanceSonographic evaluation of nerve roots and peripheral nerves may be a useful disease marker in ALS to confirm the diagnosis and to potentially monitor the disease progression.     

神经生长因子对创伤性脊髓损伤大鼠膀胱功能和轴突损伤修复的作用研究

目的 探讨神经生长因子(Nerve growth factor, NGF)对创伤性脊髓损伤(Traumatic spinal cord injury, t-SCI)大鼠膀胱功能和脊髓神经轴突损伤修复的影响及其分子机制。方法 取30只雄性Sprague-Dawley(SD)大鼠,通过改良Allen’s击打法构建创伤性脊髓损伤模型,随机分为假手术组、损伤组和NGF组,每组各10只;采用血脑屏障(Blood-brain barrier, BBB)评分观察术前、术后大鼠的后肢运动功能;BL-420生物仪实验系统检测尿动力学;甲苯胺蓝染色吻合口远端截取的左侧腰6前根,计算有髓轴突数量;采用苏木精-伊红(Hematoxylin eosin, HE)染色大鼠膀胱组织;原位末端标记法(TdT-mediated dUTP nick and labeling, TUNEL)染色大鼠损伤严重的脊髓,观察脊髓神经细胞的凋亡率;蛋白免疫印迹法(Western blot)检测脊髓组织中原癌基因丝氨酸/苏氨酸蛋白激酶(proto-oncogene serine/threonine-protein kinase, R...