正中神经电刺激对脑外伤后昏迷大鼠前额叶皮质及下丘脑Orexin-A及其受体OX1R表达变化的影响

目的:探讨正中神经电刺激(MNES)对脑外伤(TBI)昏迷大鼠的促醒作用及其可能机制。方法:将SD大鼠分为3组:空白对照组、假刺激组和刺激组。应用MNES治疗脑外伤后昏迷大鼠,观察其行为学变化,并用免疫组织化学、Western-blot、ELISA技术检测各组大鼠前额叶皮质(PFC)和下丘脑组织的Orexin-A及OX1R含量。结果:刺激组30只大鼠中21只出现翻正反射,假刺激组30只中7只出现翻正反射;将3组的Orexin-A及OX1R含量进行两两比较,发现刺激组Orexin-A及受体水平高于其他两组,差异有显著性意义(P0.05)。结论:MNES可作为脑外伤后昏迷的有效促醒手段,其机制可能与PFC及下丘脑部位Orexin-A及OX1R水平上调有关。     

迷走神经电刺激对脑外伤昏迷大鼠前额叶皮质组胺H_1受体表达的影响

目的:探讨迷走神经电刺激(vagus nerve electrical stimulation,VNS)对脑外伤(traumatic brain injury,TBI)昏迷大鼠的促醒作用及其对TBI昏迷大鼠前额叶皮层组胺H_1受体表达的影响。方法:将72只SD大鼠分为4组:空白对照组、假刺激组(TBI)、刺激组(TBI+VNS)和Orexin-1受体(OX_1R)拮抗剂组(TBI+SB334867+VNS)。应用VNS治疗脑外伤后昏迷大鼠,观察其行为学变化,并用免疫组织化学检测各组大鼠前额叶皮质组胺H_1受体(H_1R)表达的变化。结果:刺激组18只大鼠中12只出现翻正反射;假刺激组4只出现翻正反射;拮抗剂组9只出现翻正反射。免疫组化结果显示刺激组大鼠PFC区的H_1R的表达水平显著高于假刺激组和拮抗剂组(P0.05)。结论:VNS可改善脑外伤后昏迷大鼠的意识状态,有望成为TBI昏迷促醒的一种新的电刺激方法,其促醒机制可能与PFC区H_1R水平的上调有关,Orexin-A可能通过OX_1R在其中起调节作用。     

迷走神经电刺激对脑外伤后昏迷大鼠前额叶皮质去甲肾上腺素α1受体表达变化的影响

目的:探讨迷走神经电刺激(vagus nerve stimulation,VNS)对脑外伤(traumatic brain injury,TBI)后昏迷大鼠的促醒作用及其可能机制。方法:健康成年SD大鼠90只,随机分为空白对照组、假刺激组和刺激组。TBI后昏迷大鼠给予VNS治疗,观察其行为学变化,并通过免疫组织化学和Western-blot技术检测各实验组大鼠前额叶皮质(prefrontal cortex,PFC)组织中去甲肾上腺素α1受体(α1R)含量。结果:假刺激组和刺激组大鼠各30只,观察行为学变化发现,刺激组中20只出现翻正反射,假刺激组仅8只出现翻正反射;将3个实验组中α1R含量进行两两比较,发现刺激组α1R水平均显著高于其他两组(P0.05)。结论:VNS可改善TBI后昏迷大鼠的意识状态水平,对促进TBI后昏迷大鼠的觉醒有积极的治疗作用,其机制可能与PFC去甲肾上腺素α1R水平上调有关。     

丘脑电刺激对脑外伤后昏迷大鼠前额叶皮质区去甲肾上腺素α1受体表达水平的影响

目的 探讨丘脑电刺激对脑外伤后昏迷大鼠的促醒作用及其可能机制。 方法 选取健康成年Wistar种系大鼠45只，随机分为空白对照组、假刺激组和刺激组，每组15只，观察其行为学变化，并通过免疫荧光和Western blot技术检测各组大鼠前额叶皮质组织中去甲肾上腺素α1受体（α1R）的含量。 结果 刺激组中 15 只大鼠全部出现翻正反射，假刺激组仅 8 只大鼠出现翻正反射；将各组大鼠前额叶皮质区α1R含量进行比较，发现刺激组α1R含量水平显著高于假刺激组（P<0.05）。 结论 丘脑电刺激可改善脑外伤后昏迷大鼠的意识状态水平，对促进脑外伤后昏迷大鼠觉醒有积极作用，其机制可能与前额叶皮质去甲肾上腺素α1R表达水平上调有关。     

迷走神经电刺激对脑外伤昏迷大鼠意识及前额叶皮质γ-氨基丁酸b1受体表达的影响

目的研究迷走神经电刺激(VNS)对脑外伤(TBI)昏迷大鼠的促醒效果及其机制。方法 168只健康Sprague-Dawley大鼠随机分为空白组、TBI组、拮抗剂组和VNS组,每组42只。空白组不做任何处理,TBI组、VNS组、拮抗剂组撞击法复制脑外伤模型,造模后昏迷至少30 min的大鼠入选。VNS组予VNS,拮抗剂组侧脑室注射Orexin A受体1拮抗剂SB334867后加VNS,TBI组予假VNS。分别于干预后6 h、12 h、24 h观察大鼠意识水平,应用免疫组化、Western blotting检测前额叶皮质γ-氨基丁酸b1受体(GABAb1R)表达水平。结果最终空白组42只、TBI组11只、拮抗剂组13只、VNS组28只大鼠觉醒。Western blotting显示,干预后12 h、24 h,GABAb1R蛋白表达TBI组拮抗剂组空白组VNS组(F60.412,P0.001)。免疫组化显示,GABAb1R蛋白表达TBI组拮抗剂组VNS组空白组(H=15.121,P=0.002),但不同时间点无显著性差异(H=3.028,P=0.220)。结论 VNS可促TBI昏迷大鼠觉醒,其促醒机制可能与Orexin A介导大鼠前额叶皮质GABAb1R表达水平下调有关。     

深部脑刺激对脑外伤昏迷大鼠前额叶皮质γ-氨基丁酸b受体表达变化的影响

目的:研究深部脑刺激(DBS)对脑外伤(TBI)昏迷大鼠前额叶皮层γ-氨基丁酸(GABA)b受体表达变化的影响及促醒可能机制。方法:将54只成年标准大鼠随机分为空白组、假刺激组、刺激组,应用DBS治疗TBI昏迷大鼠,观察其行为学变化,实验完成后12h处死每组大鼠,用蛋白质印迹法(WB)及免疫荧光技术检测前额叶皮质区GABA b受体的表达。结果:①刺激组18只大鼠中16只大鼠出现翻正反射,假刺激组18只中9只出现翻正反射;②WB检测显示,刺激组大鼠前额叶皮质区GABA b受体表达水平显著低于假刺激组(P0.05);③免疫荧光检测显示,刺激组大鼠前额叶皮质区GABA b受体表达水平显著低于假刺激组(P0.05)。结论:DBS可提高TBI昏迷大鼠意识状态,其机制可能与下调前额叶皮质区GABA b受体表达有关。     

正中神经电刺激脑外伤昏迷大鼠前额叶皮质5-HT 2A受体表达的实验研究

目的:研究正中神经电刺激对脑外伤昏迷大鼠前额叶皮质5-羟色胺(5-HT)2A受体表达的影响。方法:将72只SD大鼠随机分为空白组、假刺激组、刺激组和拮抗剂组共4组。采用经典的自由落体模型建立脑外伤昏迷模型及大鼠意识状态6级评定标准评估四组大鼠行为学变化,并于实验完成后6、12、24h用免疫组化技术检测各组大鼠前额叶皮质组织的5-HT 2A受体表达。结果:刺激组13只大鼠(72.22%)出现翻正反射,拮抗剂组9只(50%)、假刺激组5只(27.78%)出现翻正反射。空白对照组意识状态分级秩均值为9.50级,假刺激组为52.75级,刺激组为37.61级,拮抗剂为46.14级,四组的意识状态分级呈"空刺拮假"递增趋势,组间比较差异有显著性意义(P0.01)。免疫组化结果显示组间比较前额叶5-HT 2A受体表达呈"空假拮刺"递增趋势(P0.05),组内比较呈"6h24h12h"趋势(P0.05)。结论:正中神经电刺激可作为脑外伤后昏迷的一种有效促醒手段,其机制可能与前额叶皮质5-HT 2A受体水平上调有关,且Orexin-A可能在其中发挥调节作用。     

正中神经电刺激对脑外伤昏迷大鼠前额叶皮质H1受体表达的影响

目的观察正中神经电刺激对脑外伤昏迷大鼠前额叶皮质H1受体表达的影响。 方法采用随机数字表法将72只SD大鼠分为空白对照组、假刺激组、刺激组及拮抗剂组。采用经典自由落体撞击法将假刺激组、刺激组及拮抗剂组大鼠制成脑外伤昏迷模型,刺激组大鼠于制模结束后给予正中神经电刺激,拮抗剂组于制模结束后向侧脑室注射OXR1拮抗剂并给予正中神经电刺激,假刺激组实验操作与刺激组相同,但干预期间电流强度为0。待实验结束1h后采用双盲法评定大鼠意识状态,于实验结束6h,12h及24h时采用免疫组织化学技术检测各组大鼠前额叶皮质H1受体表达情况。 结果实验结束1h后空白对照组18只大鼠意识状态均为Ⅰ级,刺激组有13只大鼠(72.2%)出现翻正反射,拮抗剂组有9只(50.0%)、假刺激组有5只(27.8%)大鼠出现翻正反射,4组大鼠意识状态组间差异均具有统计学意义(P<0.05)。免疫组化检测发现空白对照组、假刺激组、拮抗剂组及刺激组大鼠H1受体表达呈现逐渐增强态势(P<0.05);实验结束12h,6h及24h时各组大鼠H1受体表达在上述时间点呈现逐渐增强趋势,但各相邻时间点间差异无统计学意义(P>0.05)。 结论正中神经电刺激可作为脑外伤后昏迷的一种有效促醒手段,其治疗机制可能与上调前额叶皮质区H1受体表达有关,且Orexin-A可能在其中发挥调节作用。     

经颅直流电刺激对脑外伤昏迷大鼠意识及脑源性生长因子表达的影响

目的:探讨经颅直流电刺激(tDCS)对脑外伤昏迷大鼠的促醒作用及其可能机制。方法:成年SD大鼠54只,随机分为3组,每组18只,分别是空白组、脑外伤(TBI)昏迷组及tDCS组。TBI和tDCS组制作脑外伤昏迷模型,前者进行假tDCS治疗,后者用tDCS治疗。各组处理后在6h、12h、24h点评定大鼠意识水平,对比治疗前后意识水平变化,处死大鼠,获取前额叶和海马,应用Western Blot法测定大鼠组织中脑源性生长因子(BDNF)的表达。结果:空白组18只、TBI组6只、tDCS组11只出现翻正反射,TBI组大鼠前额叶与海马BDNF表达较空白组升高,在12h点,tDCS组大鼠前额叶BDNF表达较TBI组升高,差异有显著性意义(P0.05),在6h点,tDCS组大鼠海马BDNF表达较TBI组升高,差异有显著性意义(P0.05)。结论:tDCS可促TBI昏迷大鼠觉醒,其机制可能与上调前额叶及海马BDNF表达有关。     

迷走神经电刺激对脑外伤昏迷大鼠前额叶皮质5-羟色胺2A受体表达的影响

目的探讨迷走神经电刺激对脑外伤昏迷大鼠促醒作用及对前额叶皮质5-HT2A受体表达的影响。方法将72只Sprague-Dawley大鼠随机分为空白对照组、假刺激组、刺激组和拮抗剂组,每组18只。应用自由落体撞击法建立脑外伤昏迷模型。拮抗剂组注入OXR1受体拮抗剂SB334867,采用迷走神经电刺激治疗刺激组和拮抗剂组。观察其行为学变化,并用免疫组织化学技术检测各组大鼠前额叶皮质5-HT2A受体表达的影响。结果刺激组12只苏醒,拮抗剂组9只苏醒,假刺激组4只苏醒。5-HT2A受体含量从低到高依次为:空白对照组、拮抗剂组、假刺激组、刺激组(χ~2=11.464,P=0.009)。结论迷走神经电刺激可提高脑外伤昏迷大鼠意识状态,其机制可能与上调5-HT2A受体有关。     

Distribution of methylphenidate and p-hydroxymethylphenidate in rats

Rats were administered threo-dl-methylphenidate (MPH) i.v., i.p. and p.o. A gas chromatography-mass spectrometry method incorporating deuterated internal standards was used to quantify MPH and the metabolite threo-dl-p-hydroxymethylphenidate (HOMPH). Accumulation and decay of MPH were evaluated in serum and brain to assess whether serum MPH correlated with brain content. HOMPH was quantified to appraise its contribution to the central action of MPH. MPH accumulated in the brain at a concentration of 3.75 microgram/g tissue within 1 min after i.v. injection of 1 mg/kg, and averaged 8 times the concentration in serum over the first 30 min. The steady-state volume of distribution was 1.47 liters/kg. The decay of MPH from brain and serum occurred biphasically with postdistribution (beta) T1/2 values of 20 and 50 min, respectively. After p.o. administration (1 mg/kg), brain MPH concentrations also exceeded those in serum. HOMPH was detected only in liver and kidney after 1 mg/kg MPH (i.v. and p.o.). Thirty minutes after 20 mg/kg of MPH (i.p.), MPH accumulated in kidney greater than lung greater than brain greater than heart greater than liver. Brain MPH concentration was 900 times that of HOMPH (0.02 microgram/g) after this dose. In liver, conjugated HOMPH was present (12.1 microgram/g). After i.v. administration of HOMPH, less localization occurred in brain than occurred for MPH. These data suggest that the central effects of MPH do not depend upon conversion to HOMPH.     

Direct and acute cardiotoxic effects of ultrafine air pollutants in spontaneously hypertensive rats and Wistar--Kyoto rats

It is hypothesized that preexisting cardiovascular disease could affect the susceptibility to direct and acute cardiotoxic effects of ultrafine air pollutants. Ultrafine particles (UFP) isolated from 12.5 mg of diesel particulate matter (National Institute of Standards and Technology) were infused into isolated Langendorffperfused hearts obtained from spontaneously hypertensive rats (SHR) and normotensive control Wistar- Kyoto rats (WKY). Perfusion for 30 minutes with UFP reduced cardiac function in both groups-but to a greater extent in WKY. In SHR, developed pressure was reduced by 24.1 +/- 4.4% of baseline and maximal dP/dt was reduced by 19.8 +/- 4.9%; in WKY, developed pressure was reduced by 43.5 +/- 7.3% and maximal dP/dt by 41.8 +/- 8.2% (P < .05 for maximal dP/dt in SHR vs WKY). Coronary flow was decreased by 30.3% versus 53.7% in SHR versus WKY ( P < .05). The results of this study suggest that although UFP depress myocardial contractile response and coronary flow in both SHR and WKY the underlying hypertension does not necessarily worsen the response.     

Doxycycline and tissue repair in rats

Iterations in collagen turnover are integral to tissue repair. Repair gone awry, as a result of excess collagen accumulation or degradation, can contribute to pathologic ventricular remodeling. Pharmacologic interventions that would attenuate either aspect of faulty repair have therefore attracted interest. Tetracyclines, which inhibit both collagen synthesis and degradation, as well as angiogenesis, may hold promise, unrelated to their antimicrobial properties, in this regard. Assessment of their potential in rodent hearts with experimental injury can be problematic, given the often microscopic nature of tissue repair and brief involvement of matrix metalloproteinases (MMPs). We therefore selected a subcutaneous model in which granulation and fibrous tissues form over several weeks in response to croton oil and where fibrous tissue is subsequently resorbed because of high levels of collagenolytic activity. Untreated rats were compared with those given daily oral doxycycline (40 mg/kg). We harvested pouch tissue and exudate weekly for 5 weeks to assess hydroxyproline concentration and MMP activity (gelatin substrate zymography) of pouch wall and mononuclear cell count of pouch exudate. At week 2, neovascularization in pouch wall was measured by means of intravenous infusion of carmine-red dye in gelatin. The resultant "vascular cast" was solubilized and dye content quantitated with the use of spectrophotometry. Serum was assayed weekly for type I collagen carboxyterminal telopeptide (ICTP), a marker of collagen degradation. During weeks 1 and 2 and compared with untreated controls, doxycycline-treated rats had attenuated pouch tissue weight, collagen concentration, MMP2 lytic activity and vascularity, and reduced exudate volume and mononuclear cells. In vitro, doxycycline inhibited tissue gelatinolytic activity in a dose-dependent manner. At weeks 4 and 5, pouches were larger and collagen concentration was higher in doxycycline-treated rats, and serum ICTP levels were reduced at weeks 3 and 4. During the initial phase of pouch development, doxycycline exerts an inhibitory effect on tissue formation, likely mediated through its attenuation of angiogenesis and modulations of collagen turnover. As repair proceeds in subsequent weeks, doxycycline retards collagen degradation and pouch resorption by inhibiting MMPs. Doxycycline offers a multifaceted pharmacologic profile with which to modify various aspects of tissue repair in the rat.     

细胞因子在大鼠骨质疏松及疼痛中作用机制

目的 探讨去卵巢大鼠骨骼局部肿瘤坏死因子(tumor necrosis factor,TNF)-α、白细胞介素(interleukin,IL)-1β、IL-6与骨质疏松形成的关系.方法 3个月龄雌性SD大鼠80只,随机分为实验组和对照组各40只,实验组大鼠去除双侧卵巢,对照组不作处理,均于12周后处死.2组采用单光子骨矿测定仪测定大鼠椎体、椎间盘、小关节骨密度值,组织学观察骨小梁及周边成骨细胞、破骨细胞数目;采用免疫组织化学法检测骨组织中TNF-α、IL-1β、IL-6水平,并进行2组间比较.结果 实验组大鼠椎体、椎间盘、小关节局部骨密度值分别为(0.181±0.018)、(0.181±0.018)、(0.121±0.014)g/cm2,均低于对照组((0.204±0.021)、(0.205±0.022)、(0.148±0.019)g/cm2)(P＜0.05);实验组骨小梁、成骨细胞、破骨细胞占视野面积百分比分别为(6.6±0.4)％、(83.3±6.9)％、(14.1±0.5)％,与对照组((12.0±0.5)％、(98.5±7.3)％、(7.6±0.4)％)比较差异有统计学意义(P＜0.05);实验组骨组织细胞中IL-6、IL-1β、TNF-α水平分别为(158.980±0.319)、(151.560±3.314)、(147.890±2.184)μg/L,均高于对照组((156.170±0.294)、(146.250±6.900)、(145.260±1.563)μg/L)(P＜0.05).结论 去卵巢大鼠骨组织中破骨细胞比例增高,骨小梁、成骨细胞减少,破骨细胞IL-6、IL-1β、TNF-α细胞因子表达水平升高可导致骨质疏松.     

去势与非去势不同月龄雌性大鼠成骨细胞内雌激素受体β的表达特点

目的:观察去势与非去势雌性大鼠成骨细胞内雌激素受体β的表达水平,并比较其变化特点。方法:实验于2003-02/06在哈尔滨医科大学第二临床医学院科研中心完成。分别取新生,2,4,6,8,10,12个月龄的雌性Wistar大鼠各14只,按月龄分为6组,2,4,6,8,10,12个月龄组,各月龄组再随机分对照组,正常饲养不造模;去势组行卵巢切除造模,每组各7只。两个月后处死。①成骨细胞的生物学特征检测:取新生雌性Wistar大鼠14只用于大鼠成骨细胞体外培养。采用二次酶消化法、反复贴壁法分离纯化培养大鼠成骨细胞。采用倒置显微镜观察成骨细胞形态及超微结构。大鼠成骨细胞检验学特征碱性磷酸酶检测采用改良钙钴法,阳性反应为胞质中出现灰黑至深黑颗粒状或片状沉淀。成骨细胞培养上清液碱性磷酸酶及骨钙素检测,采用磷酸对硝基苯酯二钠盐比色法进行碱性磷酸酶检测,测得A值,换算成国际单位。采用放射免疫试剂盒进行骨钙素检测,计数器测定沉淀物cpm值,根据标准曲线得到样品中骨钙素含量。②成骨细胞内雌激素受体检测:应用半定量蛋白印迹酶促底物染色法以及发光法。测得A值,以A值越高,说明雌激素受体β表达越多。结果:纳入84只大鼠分为6组,均进入结果分析。①成骨细胞形态学观察:随着培养时间的延长,细胞伸出较多突起,细胞融合成片,培养12～18d呈多层生长。②大鼠成骨细胞碱性磷酸酶染色结果:镜下见90%的细胞呈碱性磷酸酶染色阳性。③成骨细胞培养上清液碱性磷酸酶及骨钙素含量:成骨细胞碱性磷酸酶和骨钙素明显高于成纤维细胞(P<0.01)。④去势与非去势大鼠成骨细胞内雌激素受体β的表达结果:应用酶促低物染色法测定,非去势组雌激素受体β表达在2个月龄组呈低度表达(90.81±2.20),4,6个月组雌激素受体β开始呈现上升趋势(121.10±5.78,143.20±4.41)至8,10个月组雌激素受体β达到高峰(186.10±6.60,190.90±6.10),10月组以后表达呈现下降趋势;去势组2,4,6,8,10,12个月组雌激素受体β都呈低度表达(87.80±2.51,89.50±2.72,89.81±2.43,90.59±2.34,92.52±2.62,90.39±2.53),除2个月龄组以外,其他各月龄组非去势组均显著高于去势组(P<0.05,0.01)。结论:①本实验采用酶消化法培养的细胞符合成骨细胞的生物学特征,具备成骨细胞功能。②去势大鼠在观察的不同时限点均出现成骨细胞雌激素受体β表达较同月龄非去势大鼠显著减少,说明去势影响成骨细胞内雌激素受体β的表达。     

Fluid reabsorption in Henle''s loop and urinary excretion of sodium and water in normal rats and rats with chronic hypertension

The function of the short loops of Henle was investigated by micropuncture technique in normal rats, in rats with spontaneous hypertension, and in the untouched kidney of rats with experimental renal hypertension. All animals received a standard infusion of 1.2 ml of isotonic saline per hr.With increasing arterial blood pressure (range from 90 to 220 mm Hg), a continuous decrease in transit time of Lissamine green through Henle's loop from 32 to 10 sec was observed. Fractional water reabsorption along the loop declined progressively from 26 to 10%, and fractional sodium reabsorption decreased from 40 to 36% of the filtered load. The fluid volume in Henle's loop calculated from transit time and mean flow rate also decreased with increasing blood pressure. There was no change in superficial single nephron filtration rate but there was a slight increase in total glomerular filtration rate (GFR). Sodium and water reabsorption in the proximal tubule remained unchanged.Urine flow rate, sodium excretion, osmolar clearance, and negative free water clearance increased with increasing blood pressure. The osmolal urine to plasma (U/P) ratio declined but did not fall below a value of 1.5. It is concluded that the increase in sodium and water excretion with chronic elevation of arterial blood pressure is caused by a decrease of sodium and water reabsorption along the loop of Henle, presumably as a consequence of increased medullary blood pressure.     

去势与非去势不同月龄雌性大鼠成骨细胞内雌激素受体β的表达特点

目的：观察去势与非去势雌性大鼠成骨细胞内雌激素受体β的表达水平，并比较其变化特点。方法：实验于2003—02／06在哈尔滨医科大学第二临床医学院科研中心完成。分别取新生，2，4，6，8，10，12个月龄的雌性Wistar大鼠各14只，按月龄分为6组，2，4，6，8，10，12个月龄组，各月龄组再随机分对照组，正常饲养不造模；去势组行卵巢切除造模，每组各7只。两个月后处死。①成骨细胞的生物学特征检测：取新生雌性Wistar大鼠14只用于大鼠成骨细胞体外培养。采用二次酶消化法、反复贴壁法分离纯化培养大鼠成骨细胞。采用倒置显微镜观察成骨细胞形态及超微结构。大鼠成骨细胞检验学特征碱性磷酸酶检测采用改良钙钴法，阳性反应为胞质中出现灰黑至深黑颗粒状或片状沉淀。成骨细胞培养上清液碱性磷酸酶及骨钙素检测，采用磷酸对硝基苯酯二钠盐比色法进行碱性磷酸酶检测，测得A值，换算成国际单位。采用放射免疫试剂盒进行骨钙素检测，计数器测定沉淀物cpm值，根据标准曲线得到样品中骨钙素含量。②成骨细胞内雌激素受体检测：应用半定量蛋白印迹酶促底物染色法以及发光法。测得A值，以A值越高，说明雌激素受体β表达越多。结果：纳入84只大鼠分为6组，均进入结果分析。①成骨细胞形态学观察：随着培养时间的延长，细胞伸出较多突起，细胞融合成片，培养12-18d呈多层生长。②大鼠成骨细胞碱性磷酸酶染色结果：镜下见90％的细胞呈碱性磷酸酶染色阳性。③成骨细胞培养上清液碱性磷酸酶及骨钙素含量：成骨细胞碱性磷酸酶和骨钙素明显高于成纤维细胞（P〈0．01）。④去势与非去势大鼠成骨细胞内雌激素受体β的表达结果：应用酶促低物染色法测定，非去势组雌激素受体β表达在2个月龄组呈低度表达（90．81．2．20），4，6个月组雌激素受体β开始呈现上升趋势（121．10&;#177;5．78，143．20&;#177;4．41）至8，10个月组雌激素受体β达到高峰（186．10&;#177;6．60，190．90&;#177;6．10），10月组以后表达呈现下降趋势；去势组2，4，6，8，10，12个月组雌激素受体β都呈低度表达（87．80&;#177;2．51，89．50&;#177;2．72，89．81&;#177;2．43，90．59&;#177;2．34,92．52&;#177;2．62，90．39&;#177;2．53），除2个月龄组以外，其他各月龄组非去势组均显著高于去势组（P〈0．05,0．01）。结论：①本实验采用酶消化法培养的细胞符合成骨细胞的生物学特征，具备成骨细胞功能。②去势大鼠在观察的不同时限点均出现成骨细胞雌激素受体β表达较同月龄非去势大鼠显著减少，说明去势影响成骨细胞内雌激素受体β的表达。     

Antithrombotic and thrombolytic activity of sulodexide in rats

Summary We evaluated the ability of sulodexide, an extracted glycosaminoglycan, to prevent thrombus formation and to reduce a stabilized thrombus in a rat venous thrombosis model (vena cava ligature). Injection of sulodexide 10 min before induction of venous stasis, prevented thrombus formation in a dose-dependent manner (median effective dose 0.55 mg/kg). When given to rats with 6-h-old thrombi, sulodexide caused a marked reduction in thrombus size which reached 70% after 2 h with the highest dose tested (2 mg/kg). The effect of sulodexide on established thrombi appears to be due, at least in part, to a fibrinolysis-mediated mechanism, since it was significantly inhibited by ε-aminocaproic acid, a well-known antifibrinolytic drug. Treatment with sulodexide did not noticeably affect plasma levels of plasminogen activator and its specific inhibitor. We also showed that fluorescein-labelled sulodexide, when given to animals with 6-h-old thrombi, was present within the thrombi harvested 2 h later, but was then absent from blood. The fluorescence was mainly located in areas filled with amorphous material, that was identified as fibrin by staining with phosphotungstic acid-hematoxylin. No fluoresceinlabelled material could be detected in rats treated with fluorescein alone. These findings indicate that, besides preventing venous thrombus formation, sulodexide is able to promote thrombus dissolution by a mechanism that is partly related to local fibrinolysis stimulation.     

Use of multiple fluorophores for evaluating microvascular permeability in control rats and rats with sepsis

Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n=11) and post-CLP (caecal ligation and puncture; n=14) Sprague-Dawley rats were injected with tracer amounts of albumin and PEG-Alb [albumin covalently linked to methoxy-poly(ethylene glycol)] labelled with fluorescein and Texas Red. Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined. The relative retention of the PEG-Alb and albumin was assessed via measurement of the TER (transcapillary escape rate; in %/h) and the t(50%) estimate, defined as the time when the actual concentration reached 50% of its baseline. The concentration-time trends for both albumin and PEG-Alb tracers exhibited two-compartmental behaviour and were analysed using bi-exponential modelling. Retention times were significantly greater for PEG-Alb in both control and CLP rats. TER(PEG-Alb) was significantly lower than TER(albumin) for both control (8.1+/-5.6 compared with 14.8+/-7.1 %/h respectively; P<0.01) and CLP (14.8+/-6.6 compared with 22.5+/-7.3 %/h respectively; P<0.001) rats. The t(50%[PEG-Alb]) was substantially greater than the corresponding t(50%[albumin]) for both control (29.8+/-9.8 compared with 7.2+/-2.0 h respectively; P<0.001) and CLP (12.9+/-5.6 compared with 5.1+/-1.6 h respectively; P<0.001) rats. The result was similar irrespective of the fluorophore-protein combination, validating the multifluorophore technique. In conclusion, the double-fluorophore approach described in the present study may provide the future basis for a method to quantify capillary leak in disease.