大鼠脊髓损伤过程中细胞周期蛋白H的表达变化

目的 观察细胞周期蛋白H(Cyclin H)在急性脊髓损伤后的表达变化及定位情况,探讨其在脊髓损伤与修复过程中的生物学功能.方法 取56只成年SD大鼠,随机分8组:正常对照组、T9撞击伤6 h、12 h、1 d、3 d、5 d、7 d、14 d 7组,每组7只.采用Western blot测定损伤后各时间段Cyclin H蛋白水平在脊髓中的表达变化,采用免疫组织化学方法检测Cyclin H在正常及损伤后脊髓中的分布和定位,在此基础上探讨Cyclin H在脊髓损伤后的病理生理意义.结果 Western blot结果表明创伤性脊髓损伤后脊髓中Cyclin H表达呈现逐渐升高再下降的趋势,3 d达到高峰.免疫组织化学表明Cyclin H在正常脊髓中表达,损伤后3 d,Cyclin H在脊髓白质中的表达明显增强,在灰质中的表达也有一定程度的增强.免疫荧光双标记表明Cyclin H与神经元标记物NeuN、小胶质细胞标记物CD11b有明显共定位,与星形胶质细胞标记物GFAP有少量共定位.结论 脊髓损伤后Cyclin H蛋白水平有明显时相改变,且与神经元、小胶质细胞和星形胶质细胞有共定位,提示Cyclin H在脊髓损伤后可能发挥了作用.     

NUMBL在大鼠脊髓损伤过程中的表达变化

[目的]观察NUMBL在大鼠脊髓损伤过程中的表达情况,探讨其在脊髓损伤和修复过程中的生物学功能.[方法]制作脊髓损伤模型,利用western blot、免疫组织化学、免疫荧光等技术观察NUMBL在损伤脊髓中的表达变化、组织分布与细胞定位,在此基础上探讨NUMBL在脊髓损伤后的病理生理意义.[结果]western blot结果显示NUMBL经历了在损伤后8 h和1 d的显著增加后开始下降,直到损伤后2周仍没有明显上调的变化趋势.免疫组化结果显示在脊髓损伤后1 d,NUMBL的表达在临近损伤中心的脊髓中显著增加,而在脊髓损伤后1周,NUMBL的表达较1d时明显降低.免疫荧光结果显示NUMBL与神经元和少突胶质细胞存在共定位,而与星形胶质细胞无明显共定位.[结论]脊髓损伤后脊髓中NUMBL的表达上调参与脊髓损伤的病理过程.     

成年脊髓损伤大鼠脊髓神经干细胞的体外培养及分化研究

目的 探讨大鼠脊髓损伤后脊髓神经干细胞的分离培养方法及分化情况.方法 采用Allen法制作大鼠脊髓损伤模型,利用无血清培养和单细胞克隆技术在成年脊髓损伤7 d大鼠脊髓中分离具有单细胞克隆能力的神经干细胞,并进行培养鉴定.结果 从成年脊髓损伤7 d大鼠脊髓中成功分离出神经干细胞,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原.分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原.结论 致伤7 d的成年大鼠脊髓组织体外町培养出神经十细胞,并分化为神经无细胞、星形胶质细胞和少突胶质细胞,有可能参与脊髓损伤的修复过程.     

脊髓损伤后促红细胞生成素对bcl-2的影响

目的 探讨促红细胞生成素（EPO）对大鼠脊髓损伤后伤区脊髓细胞凋亡和神经功能恢复的影响。方法 Wistar大鼠210只，随机分为4组：假手术组、脊髓损伤组、脊髓损伤加重组人EPO治疗组、脊髓损伤加生理盐水治疗组。采用原位末端脱氧核糖核苷酸转移酶介导dUTP标记（TUNEL标记法）检测神经元和少突胶质细胞凋亡，Western blot免疫印迹法和免疫组化染色检测bcl-2表达，免疫组化染色和图像分析方法观察对白质内神经纤维（NF-200染色）的保护作用，通过感觉诱发电位（SSEP）、运动诱发电位（MEP）和大鼠BBB后肢运动功能评分，观察损伤脊髓传导功能的恢复。结果 EPO保护组bcl-2在各时相点的表达明显增高，8h和7d时神经元和少突胶质细胞的TUNEL阳性细胞数明显减少；在7d时白质中NF-200阳性神经纤维明显增多；SSEP和MEP的平均潜伏期和波幅以及BBB功能评分明显提高，与损伤组和生理盐水治疗相比，差异有统计学意义（P〈0．01）。结论 EPO通过上调bcl-2的表达，在抑制脊髓损伤后神经元和少突胶质细胞的凋亡中起到神经保护作用。     

脊髓半切伤后胶质纤维酸性蛋白的表达及意义

目的：研究脊髓半切伤(hSCI)后脊髓远端组织星形胶质细胞中胶质纤维酸性蛋白(GFAP)表达的意义，并探讨反应性胶质化在脊髓半切伤中的作用。方法：SD大鼠25只，随机分为5组(n＝5)正常对照组、伤后1、4、7、14d组，用免疫组化及图像分析方法观察星形胶质细胞中GFAP的表达；用大鼠综合性为评分(CBS)方法对各组评分。结果：hSCI后远端星形胶质细胞GFAP表达比对照组明显增高(P＜0．01);l-14d呈进行性增高，损伤各组CBS1—14d呈降低趋势，两指标有显著相关性(r＝—0．05，P＜0．01)。结论：hSCI后星形胶质细胞通过其反应形胶质化对脊髓再生和修复其重要作用。     

大鼠脊髓损伤后胶质细胞生长因子的mRNA表达变化

目的：探讨胶质细胞牛长凼子（glial growth factor，GGF）在大鼠脊髓损伤后的表达变化及意义。方法：采川改良Allen’s脊髓损伤打击模型，用逆转录聚合酶链式反应法（RT-PCR）检测胚胎大鼠脊髓内、正常成年大鼠脊髓内以及脊髓损伤后不同时间点伤段脊髓组织中GGF的mRNA表达结果：GGF在成年大鼠正常脊髓内低水平表达，在胚胎脊髓中高水平表达。大鼠脊髓损伤后GGF的mRNA表达持续降低，在脊髓损伤后5d达到最低峰，之后GGF的mRNA表达可逐渐恢复。结论：GGF在脊髓乍长发育中起重要作用：大鼠脊髓损伤后GGF的表达降低可能与脊髓损伤后少突胶质细胞大量死亡或凋亡有关．与脊髓损伤后轴突无法再生、脱髓鞘的髓鞘再生困难有关。     

脊髓半切伤后胶质纤维酸性蛋白的表达及意义

目的:研究脊髓半切伤(hSCI)后脊髓远端组织星形胶质细胞中胶质纤维酸性蛋白(GFAP)表达的意义,并探讨反应性胶质化在脊髓半切伤中的作用。方法:SD大鼠25只,随机分为5组(n=5),正常对照组、脊髓半切伤后1d、4d、7d和14d组。用免疫组化及图像分析方法观察星形胶质细胞中GFAP的表达;用大鼠综合行为评分(CBS)对各组评分。结果:hSCI后远端星形胶质细胞GFAP表达比对照组明显增高(P<0.01);1～14d呈进行性增高,损伤各组CBS1～14d呈降低趋势,两指标有显著性相关性(r=-0.05,P<0.01)。结论:hSCI后星形胶质细胞通过其反应性胶质化对脊髓再生和修复起重要作用。     

导向因子Slit2在大鼠脊髓损伤后的表达及其意义

[目的]探讨轴突导向因子Slit2在大鼠脊髓损伤后的表达变化及其意义。[方法]Wistar雄性大鼠72只,实验分组A组(脊髓损伤组),36只;B组(假手术组),24只;C组(正常对照组),6只。A组大鼠麻醉后在无菌条件下打开椎板,以尖刀横断T10节段脊髓,以鼠尾痉挛性摆动、双下肢瘫为损伤标准;B组仅打开椎板显露硬脊膜,未损伤脊髓;C组未作手术,为正常对照。分别于脊髓损伤后12h、1、3、5、7d麻醉下快速取出T10节段的脊髓组织;分别于术后3、5、7、14d麻醉大鼠,先后以PBS缓冲液及4%多聚甲醛行心脏灌流固定,以T10节段为中心取出长约1cm的脊髓。分别通过逆转录聚合酶链反应法(RT-PCR)和免疫组织化学方法检测Slit2基因的表达水平及部位。[结果]RT-PCR大鼠脊髓损伤后12h即可检测到Slit2mRNA的表达,表达量逐渐升高并于脊髓损伤后第3d表达量达高峰,之后逐渐呈下降趋势。免疫组织化学检测Slit2蛋白定位于星型胶质细胞和少突胶质细胞的胞浆,呈棕黄色染色,于脊髓损伤后第3d出现,阳性细胞逐渐增多,于脊髓损伤后第7d,Slit2阳性表达的细胞数达到高峰并趋于稳定,伤后第14d开始逐渐下降,呈一过性增高。[结论]作为引导轴突生长方向的重要因子,Slit2在脊髓损伤的早期的一过性表达升高提示其可能参与轴突的再生与重建。     

MAG在大鼠急性脊髓损伤中的表达意义

目的：探讨轴突生长抑制因子MAG在急性脊髓损伤中的意义。方法：建立脊髓全横断损伤模型，采用组织学和免疫荧光组织化学技术，检测35只在不同时期脊髓损伤大鼠脊髓损伤区MAG的变化。结果：损伤组脊髓损伤后第1天灰质内出现片状出血、中央区碎裂，轴突断裂及脱髓鞘改变；第3天神经元肿胀，逐渐形成空洞，胶质细胞减少；1周残存神经元减少，胶质细胞增生；3周脊髓损伤区出现囊腔样变。免疫荧光组织化学显示脊髓损伤后1天即可见到髓鞘中MAG的表达，3天后达到高峰，到3周后逐渐恢复到对照组水平。结论：MAG可能在脊髓损伤中扮演着重要作用。     

神经病理性疼痛大鼠脊髓星形胶质细胞增殖活化的变化

目的观察神经病理性疼痛大鼠脊髓星形胶质细胞增殖活化的变化。方法健康成年雄性SD大鼠48只，随机分为假手术组和手术组（n=24），慢性坐骨神经挤压损伤（CCI）前1d、CCI后1、4、7、14、28d各随机取4只大鼠，测定机械痛阈和热痛阈后立即处死大鼠，取L4，5脊髓，用免疫组化方法观察胶质纤维酸性蛋白（GFAP）表达以反映星形胶质细胞激活情况。结果CCI后1d术侧机械痛阈和热痛阈开始下降，机械痛阈CCI后7d下降至最低，热痛阈CCI后4d下降至最低，CCI后28d仍处于较低水平（P〈0．05或0．01）；手术组术侧脊髓后角GFAP表达于CCI后4d开始增加，至CIC后7d达高峰，至CCI后28d仍维持于高水平（P〈0．05）。结论脊髓星形胶质细胞的增殖活化参与神经病理性疼痛的发生和维持。     

Reduction of Plasma Fibrinogen, Immunoglobulin G, and Immunoglobulin M Concentrations by Immunoadsorption Therapy with Tryptophan and Phenylalanine Adsorbents

Abstract Immunoadsorption (1A) therapy with tryptophan (TR-350) or phenylalanine (PH-350) adsorbents has been used to reduce the concentration of serum antibodies in human lymphocyte antigen (HLA)-immunized patients. Other forms of plasma purification have been reported to reduce the level of fibrinogen, which affects the blood properties. In this study we investigated the effects of IA therapy using both adsorbents on plasma fibrinogen and immunoglobulins G and M in 13 patients (8 patients were treated with TR-350, and 5 patients were treated with PH-350). During each session 1 plasma volume (2.8 ± 0.4 L of plasma) was processed through the immunocolumn and then returned to the patient together with the blood cells. Compared with the pretreatment values, the plasma fibrinogen, IgG, and IgM concentrations were significantly reduced after IA therapy (p < 0.01 for TR-350; p < 0.04 for PH-350). There was a positive correlation between the degree of reduction of plasma proteins and the number of IA treatments given. A nonpara-metric test (Wilcoxon's signed-rank test or the Mann-Whitney test) was used for statistical analysis. We conclude from our study that IA therapy effectively lowers the plasma levels of fibrinogen, IgG, and IgM and thus can be considered a valuable alternative to other blood purification methods.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

The multiply injured child

Blunt trauma is the principal cause of childhood death in many developed countries. This review outlines the differences between adults and children with respect to resuscitation and treatment of orthopaedic injuries in a child with polytrauma. Recent advances in techniques of fracture stabilization are reported.     

Dilemma of Membrane Biocompatibility and Reuse

Abstract: Numerous articles have been published on the multiple use of dialyzers and on the effect of different reprocessing chemicals and techniques on the dialyzer biocompatibility and performance. The results often appear contradictory, especially those comparing standard biocompatibility parameters. Despite this confusion, a discerning review of the published works allows certain limited conclusions to be drawn. Reprocessing of used hemodialyzers changes the biocompatibility profile of a dialyzer as defined by the parameters complement activation. leukopenia, and cytokine release. The effect of reprocessing depends on the chemicals and reprocessing technique applied and also on the type of membrane polymer being subjected to the reprocessing procedure. Reports of pyrogenic reactions indicate that the flux of the membrane also influences how suitable it is for safe reuse. An increased risk of allergic and pyrogenic reactions appears to be associated with dialyzer reuse. Furthermore, there has been a lack of investigations into the immunologic effect of the layer of adsorbed and chemically altered proteins that remains on the inner surface of reprocessed dialyzers. We conclude that the clinical benefit of dialyzer reuse cannot be generally accepted from a biocompatibility point of view.     

The analgesic effect of racemic ketamine in patients with chronic ischemic pain due to lower extremity arteriosclerosis obliterans

Background : Ketamine in sub-dissociative doses has been shown to have analgesic and phantom-Limb pain, where conventional treatment has often failed. Chronic ischemic pain due to lower extremity arteriosclerosis obliterans often responds poorly to analgesics, and the pain-generating mechanisms are not well understood.
Methods : Eight patients with rest pain in the lower extremity due to arteriosclerosis obliterans were given sub-dissociative doses of 0.15, 0.30, or 0.45 mg/kg racemic ketamine and morphine 10 mg as a 5-min infusion on four separate days in a cross-over, double-blind, randomised protocol. Plasma levels of (S)- and (R)-ketamine and their nor-metabolites were analysed with an enantioselective high-performance liquid chromatography (HPLC) method. Pain levels were evaluated with a visual analogue scale (VAS).
Results : Individual pain levels were highly variable during and after all the infusions but the pooled pain levels showed a dose-dependent analgesic effect of ketamine with a transient but complete pain relief in all patients at the highest dose (0.45 mg/ kg). Side-effects, mainly disturbed cognition and perception, were pronounced and dose-dependent. Morphine 10 mg had an analgesic peak at 20 min and 5/8 patients had complete pain relief. The remaining 3 patients also had high baseline pain scores, indicating a higher analgesic potency for the 0.30 and 0.45 mg/ kg ketamine doses than for morphine 10 mg.
Conclusion : We have demonstrated a potent dose-dependent analgesic effect of racemic ketamine in clinical ischemic pain. Due to a narrow therapeutic window, this analgesic effect is probably best utilised in combination with other analgesics.     

Intravenous endotoxin does not increase tissue extravasation of albumin in rats

Background : It is unclear whether activation of the inducible nitric oxide synthase (iNOS) increases or decreases the extravasation of plasma.
Methods : Chloralose anaesthetised male Wistar rats received E. coli lipopolysacharide (LPS), 3 mg kg^-1 i.v., or the corresponding volume of saline, 3 or 5 h before the end of the experiment. Mean arterial pressure (MAP) and heart rate (HR) were recorded. Tissue clearance of radio-labelled albumin, during the last 2 h of each experiment, was determined by a double-isotope method. In separate animals, the serum concentration of nitrite and nitrate was determined, 5 h after LPS or the solvent.
Main Results : LPS initially decreased MAP and lastingly increased HR. In the 3-h LPS animals (n=8), tissue plasma clearance was lower in the heart and calf muscle and increased only in diaphragm, compared to corresponding control animals (n=8). In the 5-h LPS rats, clearance was lowered (n=8) in the entire gastrointestinal tract and in testes, compared to controls (n=8). The serum nitrite/nitrate concentration was higher in animals given LPS (n=6) than in controls (n=6).
Conclusion : After LPS, tissue clearance of albumin was not increased in any major tissue, in spite of increased serum levels of NO end products. Apparently, after activation of iNOS, the augmented release of NO is not necessarily associated with increased albumin extravasation.     

Adrenaline markedly improves thoracic epidural analgesia produced by a low-dose infusion of bupivacaine, fentanyl and adrenaline after major surgery: A randomised, double-blind, cross-over study with and without adrenaline

Background: Basic pharmacological research indicates that there are synergistic antinociceptive effects at the spinal cord level between adrenaline, fentanyl and bupivacaine. Our clinical experience with such a mixture in a thoracic epidural infusion after major surgery confirms this. The objectives of the present study were to evaluate the effects on postoperative pain intensity, pain relief and side effects when removing adrenaline from this triple epidural mixture. Methods: A prospective, randomised, double-blind, cross-over study was carried out in 24 patients after major thoracic or abdominal surgery. Patients with only mild pain when coughing during a titrated thoracic epidural infusion of about 10 ml · h^?1 of bupivacaine 1 mg · ml^?1, fentanyl 2 μg · ml^?1, and adrenaline 2 μg · ml^?1 were included. On the 1^st and 2^nd postoperative days each patient was given a double-blind epidural infusion, at the same rate, with or without adrenaline. The effect was observed for 4 h or until pain when coughing became unacceptable in spite of a rescue analgesic procedure. Rescue analgesia consisted of up to two epidural bolus injections per hour and i.v. morphine if necessary. All patients received rectal paracetamol 1 g, every 8 h. Fentanyl serum concentrations were measured with a radioimmunoassay technique at the start and end of each study period. Main outcome measures were extent of sensory blockade and pain intensity at rest and when coughing, evaluated by a visual analogue scale, a verbal categorical rating scale, the Prince Henry Hospital pain score, and an overall quality of pain relief score. Results: The number of hypaesthetic dermatomal segments decreased (P <0.001) and pain intensity at rest and when coughing increased (P <0.001) when adrenaline was omitted from the triple epidural mixture. This change started within the first hour after removing adrenaline. After 3 h pain intensity when coughing had increased to unacceptable levels in spite of rescue analgesia (epidural bolus injections and i.v. morphine). Within 15–20 min after restarting the triple epidural mixture with adrenaline, pain intensity was again reduced to mild pain when coughing. Serum concentration of fentanyl doubled from 0.22 to 0.45 ng · ml^?1 (P <0.01), and there was more sedation during the period without adrenaline. Conclusions: Adrenaline increases sensory block and improves the pain-relieving effect of a mixture of bupivacaine and fentanyl infused epidurally at a thoracic level after major thoracic or abdominal surgery. Serum fentanyl concentrations doubled and sedation increased when adrenaline was removed from the epidural infusion, indicating more rapid vascular absorption and systemic effects of fentanyl.