脓毒症小鼠心肌损伤与中性粒细胞浸润的关系

目的:探讨脓毒症小鼠心肌损伤与中性粒细胞浸润的关系。方法:复制脓毒症动物模型,分对照组、假手术组、造模组,并设不同的时间点(2h、4h、8h、12h)。采用双抗夹心酶联免疫法(ELISA)检测血清肌钙蛋白(IcTnI),并测心肌组织髓过氧化物酶(MPO)活性。结果:正常组与假手术组各时间点血清cTnI、心肌组织MPO活性均无显著性差异。造模组心肌组织MPO活性较正常组和假手术组均有明显升高(P<0.05),且随着时间进展而增加;造模组血清cTnI浓度随着时间进展而增加,2小时与正常组及假手术组无显著性差异(P>0.05),4小时后显著高于正常组和假手术组(P<0.05);血清cTnI浓度与心肌组织MPO活性呈显著正相关(r=0.700,P=0.000)。结论:脓毒症心肌损伤时,心肌组织存在中性粒细胞浸润,中性粒细胞浸润程度与心肌损伤显著正相关。     

对淤血再灌注肠神经组织损伤机制的探讨

目的:建立大鼠肠淤血再灌注动物模型,探讨淤血再灌注肠神经组织损伤的机制,为临床相关疾病的诊断、治疗提供理论依据.方法:成年Wistar大鼠60只,雌雄不限,随机分为正常组、对照组和实验组,每组20只.实验组采用阻断门静脉1h后开放的方法建立大鼠小肠淤血再灌注模型,对照组只进行同样腹部手术操作但不夹闭门静脉,正常组不手术.6小时后取各组下腔静脉血,测定血清中超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量,然后处死动物,取距回盲部15厘米处肠管1厘米,采用伊红-苏木素(HE)染色观察肠粘膜组织形态学变化;用免疫组织化学方法观察正常组、对照组和实验组小肠壁肠神经组织中微管相关蛋白2(MAP-2)的表达情况.结果:HE染色可见,正常组、对照组为正常肠道管壁结构,实验组肠壁各层有比较明显的淤血、出血,小肠绒毛固有层水肿,黏膜上皮有脱落、坏死;实验组MAP-2的表达明显低于正常组及对照组(P＜0.05);与正常组及对照组相比较,实验组SOD活性明显降低(P＜0.05),MDA的含量则明显升高(P＜0.05).结论:肠淤血再灌注可能导致肠道神经元数量减少,其机制可能与肠淤血再灌注造成的自由基损伤和脂质过氧化有关.     

兔下颌骨慢性化脓性骨髓炎动物模型的建立

目的 探讨下颌骨慢性化脓性骨髓炎的造模方法.方法 成年新西兰兔22只,于下颌骨体外侧近正中联合处开骨窗注入0.1 mL5％鱼肝油酸钠和0.1 mL5.0×108 CFU/mL金黄色葡萄球菌悬液,术后常规回笼饲养.于6周开始采用以下方法检测:①肉眼大体观察;②双能X线骨密度检测;③CBCT(锥束CT)放射学形态观察;④局部细菌培养;⑤组织病理学评价.结果 所有新西兰兔早期精神状况较差,手术区域肉眼观有瘘管并有脓性分泌物.双能X线骨密度检测发现手术区骨密度(BMD)值变化不具有统计学意义(P＞0.05).CBCT显示骨缺损区边缘模糊,有骨破坏迹象.局部取材细菌培养有金黄色葡萄球菌生长.病理切片显示有不同程度的淋巴细胞、中性粒细胞、桨细胞浸润,死骨形成.结论 采用兔下颌骨体外侧近正中联合处造骨缺损并注射一定量细菌悬液的方法能够获得理想的慢性化脓性骨髓炎模型.     

禽流感H5N1亚型病毒感染恒河猴模型的建立及禽流感发病机制初探

[目的]建立禽流感H5N1病毒感染恒河猴的动物模型,探讨禽流感在哺乳类动物的发病机制。[方法]通过“环甲膜穿刺术”经气管注射鸡胚培养的禽流感H5N1病毒(AF148678;ACGoose/Guangdong/11961H5N1)感染恒河猴,观察恒河猴染毒后出现的临床体征,用显微计数法检测外周血白细胞的动态变化,用ELISA检测禽流感病毒特异性抗体变化规律,用流式细胞仪检测外周血T淋巴细胞及其亚群的动态变化。在染毒后第1天、第3天、第10天和第14天分别剖杀染毒组恒河猴1只,HE染色观察主要组织器官的病理变化,用病毒分离、免疫组化和RT-PCR三种方法分析禽流感病毒侵袭机体的特点。[结果]临床症状和体征:急性起病,表现为发热,呼吸困难,精神状态下降,活动度明显减少,食欲下降,咳嗽,紫绀等,肺部听诊双肺可闻及干、湿音。1、病理特点:以肺部损伤为主,伴多器官病变。肺部的病变主要表现为弥漫性肺泡损伤,先后经历渗出期、增生期和纤维化期;在肝脏、肾脏和中枢神经系统中也观察到变性、坏死等病理变化。2、病毒侵袭机体的特点:病毒只在呼吸系统中复制,不在呼吸道以外的组织器官中复制;肺内支气管上皮细胞、肺泡上皮细胞和肺巨噬细胞是禽流感病毒侵犯的主要细胞类型。3、外周血象特点:外周血白细胞总数、淋巴细胞数出现短暂的下降,中性粒细胞数先升后降,但均于感染第7天后逐渐恢复到正常水平。4、抗体变化特点:感染后第7~11天,抗体水平持续快速升高;感染第11天后,抗体水平呈逐渐缓慢升高趋势(观察到染毒后50天为止)。5、细胞免疫特点:细胞免疫功能受损,表现为CD3+T淋巴细、CD3+CD4+T淋巴细胞和CD3+CD8+T淋巴细胞均出现短暂的下降,但这种细胞免疫功能受损是可逆的,到感染第7天后逐渐恢复回升至正常。[结论]1、恒河猴感染后的临床特点、病理变化、外周血象、免疫反应等均与人禽流感严重病例相类似,表明该模型是成功的,可为禽流感病毒在人体内致病机理的研究以及抗禽流感病毒的药物和疫苗评价提供最近似于人类的动物模型。2、综合本研究的实验结果,我们认为,H5N1禽流感毒主要攻击的对象为呼吸系统,不在呼吸道以外的组织器官中复制。禽流感病毒感染引起的急性弥漫性肺损伤是发病的中心环节,其发病可能经过病毒侵入、复制阶段,免疫损伤阶段和多器官功能损伤阶段。     

The upper esophageal sphincter in the cat: the role of central innervation assessed by transient vagal blockade

Studies were performed on four cats to assess the role of extrinsic innervation via the cervical nerve trunks in the control of upper esophageal sphincter function. Transient vagal nerve blockade was accomplished by cooling the cervical vagosympathetic nerve trunks previously isolated in skin loops on each side of the neck. Upper esophageal sphincter pressure was measured using a multilumen oval manometry tube and a rapid pull-through technique. The upper esophageal sphincter response to cervical intraesophageal balloon distention and acid perfusion was assessed. The feline upper esophageal sphincter has a distinct asymmetric pressure profile, whereby anterior pressure greater than posterior pressure greater than left pressure greater than right pressure. Bilateral vagal nerve blockade lowered the mean upper esophageal sphincter pressure from 18.5 +/- 1.5 to 12.0 +/- 2.8 mmHg (1 mmHg = 133.3 Pa) (p less than 0.001), with a significant reduction in pressure in all four quadrants. Intraesophageal balloon distention and acid perfusion both produced a significant increase in upper esophageal sphincter pressure. Bilateral vagal nerve blockade completely abolished the response of the upper esophageal sphincter to balloon distention and acid perfusion. We conclude that normal upper esophageal sphincter tone in the cat is partially mediated by excitatory neural input via the cervical nerve trunks, presumably via the recurrent laryngeal nerves; and cervical intraesophageal balloon distention and acid perfusion produce reflex contraction of the upper esophageal sphincter, which is dependent on neural pathways via the cervical vagal nerve trunks, but the relative contribution of afferent and efferent pathways remains unknown.     

Problems associated with the measurement of mean circulatory filling pressure by the atrial balloon technique in anaesthetized rats.

To examine the existence of pressure equilibrium between tributary veins and the central vena cava during the mean circulatory filling pressure manoeuvre, pressures in the hepatic portal vein, renal vein, and inferior vena cava were determined at 4-s intervals over a 20-s period of circulatory arrest induced by inflating a right atrial balloon in normal blood volume, 10% volume depletion, and 10% volume expansion states in urethane-anaesthetized rats. Portal vein pressure determined 8 s after arrest during volume depletion and expansion was significantly higher than vena caval pressure (6.2 +/- 0.8 vs. 3.4 +/- 0.2 and 7.7 +/- 0.5 vs. 6.2 +/- 0.4 mmHg (1 mmHg = 133.32 Pa), respectively; p less than 0.01); this pressure disequilibrium continued for 16 s during volume expansion and for the entire 20 s during volume depletion. Renal vein pressure was equal to vena caval pressure during this manoeuvre. Portal vein pressure at normal blood volume was not significantly different from vena caval pressure following circulatory arrest (4.6 +/- 0.3 vs. 3.8 +/- 0.4 mmHg, respectively). Following ganglionic blockade, portal vein pressure was still significantly higher than vena caval pressure for 12 s during volume alterations. At the 8th s of the arrest the portal pressure determined in volume depletion was 3.6 +/- 0.3 mmHg and the inferior vena caval pressure was 2.6 +/- 0.4 mmHg (p less than 0.05). Under the volume expansion condition, the respective values were 6.5 +/- 0.3 and 5.3 +/- 0.4 mmHg (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oral supplement of L-glutamine on diverted colon wall

Diverted colorectal segments can present trophic and inflammatory changes. These alterations are of special importance in the patients whose colostomy becomes permanent, as well as in the differential diagnosis with other inflammatory diseases. This study was accomplished to quantify these alterations and to determine if oral supplement of L-glutamine would avoid them. Twenty-six adult male Wistar rats were distributed in three groups: control, colostomized and colostomized+L-glutamine. The colostomized group received a loop colostomy. The colostomized+L-glutamine group received a colostomy similar to the previous group and oral supplement of L-glutamine. Partial volumes of all layers of the colonic wall were measured by image analysis stereology. The diversion caused a decrease of partial volumes of the mucosa and the epithelium as well, and also of the height of the intestinal crypts ( p <0.05). There was an increase of partial volumes of the lamina propria, of the submucosa and of the muscularis mucosae vs controls ( p <0.05). The partial volume of the muscularis propria didn't show significant alteration. The supplementation of L-glutamine was effective in preventing the atrophy of mucosa and epithelium ( p <0.05), also avoiding the increase of partial volumes of the submucosa and lamina propria ( p <0.05). This supplement didn't change significantly the muscular layers. In conclusion, colostomy causes the atrophy of the colon wall, mainly due to the atrophy of the epithelium. The supplementation of L-glutamine is able to avoid these changes.     

Comparison of lung proteome profiles in two rodent models of pulmonary arterial hypertension

We studied the lung proteome changes in two widely used models of pulmonary arterial hypertension (PAH): monocrotaline (MCT) injection and chronic hypoxia (CH); untreated rats were used as controls (n = 6/group). After 28 days, invasive right ventricular systolic pressure (RVSP) was measured. Lungs were immunostained for alpha-smooth muscle actin (alphaSMA). 2-DE (n = 4/group) followed by nano-LC-MS/MS was applied for protein identification. Western blotting was used additionally if possible. RVSP was significantly increased in MCT- and CH-rats (MCT 62.5 +/- 4.4 mmHg, CH 62.2 +/- 4.1 mmHg, control 25.0 +/- 1.7 mmHg, p<0.001). This was associated with an increase of alphaSMA positive vessels. In both groups, there was a significantly increased expression of proteins associated with the contractile apparatus (diphosphoHsp27 (p<0.001), Septin2 (p<0.001), F-actin capping protein (p<0.01), and tropomyosin beta (p<0.02)). In CH, proteins of the nitric oxide (Hsc70; p = 0.002), carbon monoxide (biliverdin reductase; p = 0.005), and vascular endothelial growth factor (VEGF) pathway (annexin 3; p<0.001) were significantly increased. In MCT, proteins involved in serotonin synthesis (14-3-3; p = 0.02), the enhanced unfolded protein response (ERp57; p = 0.02), and intracellular chloride channels (CLIC 1; p = 0.002) were significantly elevated. Therefore, MCT- and CH-induced vasoconstriction and remodeling seemed to be mediated via different signaling pathways. These differences should be considered in future studies using either PAH model.     

痛泻要方对肝气乘脾泄泻小鼠肠道微生物活度的影响

目的探讨痛泻要方对肝气乘脾泄泻小鼠肠道微生物活度的影响,为痛泻要方通过调节肠道微生物治疗肝气乘脾泄泻提供实验基础和理论依据。方法将25只SPF级雄性昆明小鼠随机分为正常组10只和模型组15只,造模成功后,随机选取正常组和模型组动物各5只,在无菌环境下提取肠道内容物和肠黏膜,剩余模型动物随机分为自然恢复组和治疗组各5只,治疗成功后处死各组动物提取肠道内容物和肠黏膜。用荧光素二乙酸法测定各样品中微生物活度。结果肝气乘脾泄泻造模小鼠肠道内容物微生物活度高于正常组,与正常组相比差异有统计学意义(P=0.01),而模型组小鼠肠黏膜微生物活度降低,与正常组相比差异有统计学意义(P=0.01)。痛泻要方治疗组和自然恢复组小鼠肠道内容物微生物活度均降低,与正常组相比差异无统计学意义(P>0.05),治疗组和自然恢复组小鼠的肠黏膜前段和后段均与正常组差异有统计学意义(P=0.001),肠黏膜中段自然恢复组与正常组差异有统计学意义(P=0.043),而治疗组与正常组没有差异。结论肝气乘脾泄泻造模使肠道内容物微生物活度增加,而使肠黏膜微生物活度降低。痛泻要方能够使肝气乘脾泄泻小鼠肠道内容物中微生物活度降低,恢复至正常水平,增加肠黏膜中段微生物活度,对肠黏膜后段微生物活度增加作用更显著。     

Effects of Topical Bimatoprost 0.01% and Timolol 0.5% on Circadian IOP,Blood Pressure and Perfusion Pressure in Patients with Glaucoma or Ocular Hypertension: A Randomized,Double Masked,Placebo-Controlled Clinical Trial

PurposeTo compare the 24-hour (24h) effects on intraocular pressure (IOP) and cardiovascular parameters of timolol 0.5% and bimatoprost 0.01% in open angle glaucoma and ocular hypertensive subjects.MethodsIn this prospective, randomized, double masked, crossover, clinical trial, after washout from previous medications enrolled subjects underwent 24h IOP, blood pressure (BP) and heart rate (HR) measurements and were randomized to either topical bimatoprost 0.01% at night plus placebo in the morning or to timolol 0.5% bid. After 8 weeks of treatment a second 24h assessment of IOP, BP and HR was performed and then subjects switched to the opposite treatment for additional 8 weeks when a third 24h assessment was performed. The primary endpoint was the comparison of the mean 24h IOP after each treatment. Secondary endpoints included the comparisons of IOP at each timepoint of the 24h curve and the comparison of BP, HR, ocular perfusion pressure and tolerability.ResultsMean untreated 24h IOP was 20.3 mmHg (95%CI 19.0 to 21.6). Mean 24h IOP was significantly lower after 8 weeks of treatment with bimatoprost 0.01% than after 8 weeks of treatment with timolol 0.5% bid (15.7 vs 16.8 mmHg, p = 0.0003). Mean IOP during the day hours was significantly reduced from baseline by both drugs while mean IOP during the night hours was reduced by -2.3 mmHg (p = 0.0002) by bimatoprost 0.01% plus placebo and by -1.1 mmHg by timolol 0.5% bid (p = 0.06). Timolol 0.5% significantly reduced the mean 24h systolic BP from baseline, the diastolic BP during the day hours, the HR during the night hours, and the mean 24h systolic ocular perfusion pressure.ConclusionBoth Bimatoprost 0.01% and Timolol 0.5% are effective in reducing the mean 24h IOP from an untreated baseline but Bimatoprost 0.01% is more effective than timolol 0.5% throughout the 24h. Timolol 0.5% effect on IOP is reduced during the night hours and is associated with reduced BP, HR and ocular perfusion pressure.

Trial Registration

EU Clinical Trial Register and EudraCT# 2010-024272-26     

Effects of a bradycardic agent on postischemic cardiac recovery in rabbits.

Decreasing heart rate might be beneficial for improvement of myocardial energetics and could reduce the severity of myocardial ischemia. We examined the contribution of heart rate reduction by cilobradine (DK-AH 269), a direct sinus node inhibitor, on left ventricular function and peripheral vasomotion in anesthetized rabbits with experimental myocardial infarction. The rabbits were randomized to receive either placebo (n=10) or cilobradine (n=7). Cilobradine decreased significantly heart rate from 163 +/- 33 to 131 +/- 13 bpm, p< 0.05, without any inotopic or vascular effects. After 60 min coronary occlusion and 30 min reperfusion, both systolic and diastolic ventricular function were more reduced in the cilobradine group; i.e. maximal left ventricular pressure significantly decreased to 62 +/- 11 mmHg, p < 0.05 (placebo: 77 +/- 9 mmHg); dP/dt(min) significantly decreased to -904 +/- 247 mmHg, p < 0.05 (placebo: -1106 +/- 242 mmHg). However, infarct size in the cilobradine group was significantly smaller compared with the placebo group. In conclusion, cilobradine reduced heart rate without any negative inotropic effect and reduced infarct size. On that account, this bradycardic agent might open a promising therapeutical avenue to treat postischemic dysfunction.     

Integrity of gut mucosa during anaesthesia in major abdominal surgery

The aim of the study was to examine a perfusion and integrity of small bowel in 60 subsequent patients during the major open abdominal surgery which lasted from 2 to 7 hours. Two samples of the intestinal mucosa were removed: at the beginning, and at the end of the surgical procedure in general anaesthesia. A mucosal injury was classified into 4 grades. pH, PCO2 and lactate level were measured in the blood samples from the arterial and mesenteric vein in one hour time intervals. The changes of intestinal mucosa were found in 31 patients (51.7%): in 19 patients (31.7%) grade 1 changes were recorded, in 10 patients (16.7%) grade 2, and in 2 patients (3.3%) grade 3. Grade 4 lesions were not recorded. There was a statistically significant correlation between grades of the mucosal damage and the surgery duration (p = 0.001). Analysis during the one hour intervals showed that there was no exact time point when the significant aggravation of the pathohistological changes in intestinal mucosa occurred. However, when patients were allocated into two subgroups with surgical procedures lasting less than 4 hours and more than 4 hours, there was a statistically significant difference in the grades of mucosal damage between subgroups (p < 0.05). More biopsies without pathohistological changes were observed in the patients whose procedure duration was < 4 hours. A significantly higher lactate concentrations in arterial and mesenteric venous blood were observed in the patients with pathohistological changes at 6 hours time point as compared to 2 hour time point in the patients without pathohistological changes (p < 0.05). During the open abdominal surgery in general anaesthesia, the length of the procedure influences the grade of the intestinal mucosa injury. Deterioration of the pathohistological findings in the intestinal mucosa correlates with high lactate blood level, suggesting that the cause of these changes may result from tissue hypoxia.     

内毒素血症大鼠肠道内毒素移位的研究

肠源性内毒素血症是多器官功能衰竭的重要发病因素之一,但肠源性内毒素血症的发病机理迄今尚未阐明,本工作在离体动物,离体大鼠回肠血管灌流标本和分离的小肠粘膜刷状缘微囊制备上,观察到循环血中内毒素正反馈地促进肠道内毒素移位,多种调节肽参与内毒素移位的调控,内皮素促进而心钠素抑制其移位过程,肠道内毒素的移位主要不是通过跨细胞途径,本工作还发现牛磺酸具有抑制肠道素移位,改善内毒素血症动物预后的作用,提示牛磺