Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 10¹ copies/μL and 6.15 × 10¹ copies/μL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.     

一种介入技术制作的局灶性脑栓塞溶栓实验模型

目的运用介入技术建立一种能够符合影像学研究和溶栓的局灶性脑栓塞动物模型。材料与方法13只成年家犬,采用随机数字表法分成对照组(A组)3只和实验组10只,实验组又按照导管插入颈内动脉的不同位置分成B、C组,每组各5只,经股动脉插管注入自体动脉血栓制作犬的局灶脑栓塞动物模型。每组分别行数字减影血管造影(DSA)、磁共振扩散加权成像(DWI)扫描、神经功能评分、脑组织病理切片评价效果,并做统计分析。结果A组未见异常改变。B、C组血管实时造影示颈内动脉主干栓塞,B1、B4、B5延迟造影阻塞血管显示再通,B2、B3、C组延迟造影阻塞血管无再通,B、C组模型成功率分别为40%、100%;两组神经功能评分分别为5.2±4.438、7.2±1.923;阻塞血管无再通者DWI1h均出现梗死灶;HE染色:自溶组出现大量神经元细胞坏死性改变,C组神经元细胞形态改变程度较轻,残存细胞数目明显增多。结论运用介入技术导管插入颈内动脉内制作局灶性脑栓塞溶栓实验模型,造模稳定,动物创伤小,适合溶栓实验研究。     

用McAb-AST对犬内脏利什曼病早期诊断的初步研究

本文报道实验感染杜氏利什曼原虫小犬10只,在感染前及咸感后5、12、20、40d,分别收集血清,用McAb-AST检测血清内循环抗原。试验结果可见小犬感染后5d即出现阳性反应,表明已可检测出其血清内循环抗原,并随着时间的加长,阳性反应程度有所增加。初步建立了犬利什曼病的一种新的早期诊断方法,简便易行,易于推广应用。     

心力衰竭犬心房电生理特性的研究

为观察心力衰竭 (简称心衰 )犬心房肌电生理特性的改变 ,探讨充血性心衰时心房颤动 (AF)发生机制。选择14只犬随机分为起搏组 (n =7)和假手术组 (n =7) ,在左、右房各缝植 4对电极 ,电极尾端经皮下由犬背部穿出。假手术组犬埋置起搏器后不起搏。起搏组犬置入实验用VOO型起搏器快速心室起搏 (2 2 0次 /分 ) 6周 ,建立心衰犬模型 ,分别于起搏前、起搏 6周后 ,测定心房有效不应期 (AERP)、AERP离散度 (AERPd)、房内和房间传导时间及心房肌传导速度 ,记录AF诱发情况。结果 :①假手术组犬术前与术后比较 ,心功能和心房电生理特性均无明显变化。②心室快速起搏 6周犬AERP较起搏前略延长 ,但差异无显著性。起搏 6周犬AERPd较起搏前明显增大 (4 0 .4±15 .6msvs 2 2 .6± 10 .2ms,P <0 .0 5 )。与起搏前比较 ,起搏 6周犬房内及房间传导时间明显延长 (CTRA5 4 .7± 7.2msvs 33.1± 9.5ms ;CTLA5 2 .3± 8.9msvs 31.7± 6 .3ms ;CTRA LA6 9.7± 8.2msvs 4 2 .8± 7.9ms,P均 <0 .0 5 ) ,心房肌传导速度显著减慢 (CVRA5 4 .8± 7.9cm/svs 90 .7± 8.4cm/s ;CVLA5 7.4± 9.6cm/svs 94 .6± 10 .2cm/s,P均 <0 .0 5 )。③心衰犬AF诱发率、诱发次数、AF持续时间较起搏前明显增加。假手术组犬术前、术后比较AF诱发情况无?     

犬肾去交感神经对长期快速心房起搏后心房基质和心房颤动诱发的影响

目的 探讨犬肾去交感神经对长期快速右房起搏后心房基质和心房颤动﹙AF﹚诱发的影响。方法 20 只犬分为假手术组﹙n=6﹚、右房起搏﹙RAP﹚组﹙n=7﹚和肾去交感神经﹙RSD﹚组﹙n=7﹚三组。假手术组植入起搏器后程控关闭起搏器;RSD 组先行双侧肾交感神经消融,经 6 周恢复后植入起搏器;RAP 组和 RSD 组快速起搏﹙400 ~450 次 / 分﹚右房 5 周。所有犬在试验开始时和终点进行血压测量及电生理检测;处死动物后迅速取左房组织,检测组织心房利钠肽﹙ANP﹚、血管紧张素Ⅱ﹙AngⅡ﹚和醛固酮水平,及组织半胱天冬酶-3﹙Caspase-3﹚、Bax、B 淋巴细胞瘤-2 基因﹙Bcl-2﹚、金属蛋白酶组织抑制剂-1﹙TIMP-1﹚、基质金属蛋白酶-9﹙MMP-9﹚和转化生长因子-β1﹙TGF-β1﹚等蛋白表达。结果 RSD 组犬肾动脉消融 6 周后﹙植入起搏器前﹚收缩压较消融前显著降低﹙P=0. 001﹚;右房起搏5 周后 RSD 组和 RAP 组的收缩压和舒张压较假手术组均明显降低﹙P〈0. 05﹚,且 RSD 组收缩压较 RAP 组更低﹙P =0. 036﹚。与假手术组和 RSD 组相比,RAP 组起搏 5 周后诱发的 AF 持续时间明显延长﹙P 〈0. 05﹚。RAP 组心房有效不应期﹙AERP﹚明显缩短﹙P=0. 002﹚,而 RSD 组 AERP 的缩短无显著性﹙P =0. 087﹚。与假手术组比较,RAP 组犬左房的 ANP、AngⅡ和醛固酮水平显著升高,而在 RSD 组其升高的趋势明显减缓﹙P〈0. 05﹚。与 RAP 组相比,RSD 组犬左房的 Caspase-3、Bax、MMP-9 和 TGF-β1 蛋白表达显著降低,而 Bcl-2 和 TIMP-1 蛋白表达显著升高。结论 肾去交感神经可抑制长期快速右房起搏后 AF 的诱发和心房基质改变。     

双侧无铆型房间隔缺损封堵器的实验研究

目的在犬房间隔缺损(ASD)模型上评价双侧无铆型ASD封堵器的疗效、安全性和生物相容性。方法自行设计封堵器是由镍钛合金丝编织成的网状结构,外形呈双盘状,其左房面平整,右房面中间凹陷,中央有与推送杆相接的连接袢,内衬有三层聚酯膜。犬ASD模型建立采用房间隔穿刺结合球囊扩张方法,模拟临床经导管封堵ASD过程,闭合人工制备的ASD。用心脏超声观察封堵器位置及封堵器对右、左房室瓣功能的影响。术后1、2、3、6个月分别处死动物行组织学检查,观察封堵器周围及表面的组织学反应。结果8条犬成功制作ASD模型并封堵,超声显示封堵器位置固定良好,不影响右、左房室瓣功能。各观察时间点肉眼见所有封堵器表面光滑均覆有薄层的新生内膜。3个月后封堵器表面完全内皮化,光镜和电镜检查显示封堵器置入部位周围组织无明显的炎症反应。6个月扫描电镜观察金属丝无腐蚀。HE染色显示肺、肝、脾、肾、胰等组织无血栓栓塞、炎症表现。结论双侧无铆型ASD封堵器操作简便、使用安全及有良好的生物相容性,提示自制的ASD封堵器将可安全地应用于临床。     

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.     

两种自锁托槽对非拔牙病例牙弓变化影响的对比研究

目的：通过分析两种自锁托槽治疗非拔牙病例在排齐整平阶段后牙弓宽度、长度、周长变化,比较主动与被动自锁托槽对排齐整平后牙弓变化影响的差异性。方法牙弓周长增加量为因变量Y,矫治前后各牙弓测量值的变化（WU/L3、WU/L4、WU/L5、WU/L6、U/LL）为自变量进行分析。比较主动矫治器（主动组）与被动矫治器（被动组）矫治后牙弓变化情况。再将各项测量值的变化量进行t检验,比较两种不同矫治器矫治后牙弓各项测量值变化量有无统计学意义。结果两组矫治器相关与回归结果显示牙弓周长、牙弓宽度与长度增加量有相关性,回归方程检验均显示有统计学意义（P<0.05）。主动组与被动组各测量值比较显示WU3、WL3、WL6、LR 4项测量值有统计学意义（P<0.05）。结论主动组与被动组都有良好的扩弓效应。被动组在上下颌尖牙扩展优于主动组,而主动组在下颌第一磨牙与下牙弓周长的扩展优于被动组。     

At-risk but viable myocardium in a large animal model of non ST-segment elevation acute coronary syndrome: cardiovascular magnetic resonance with ex vivo validation

Background

Patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE).

Methods

In a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing.

Results

Serum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium (53.2 ± 4.9 vs. 43.6 ± 2.8 ms, p < 0.01) with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals (0.72 ± 0.07 vs. 1.04 ± 0.07, p < 0.001). Lower RRR corresponded to higher final TnI levels (R² = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression.

Conclusions

A large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS.     

犬肝不灌洗低温保存的实验研究——Ⅱ-酶组织化学动态观察

本文是对犬肝进行不灌洗低温保存(25℃、15℃)的系列研完之二,仍采用组织化学方法。通过对与肝脏能量关系密切的琥珀酸脱氢酶和乳酸脱氢酶动态观察,进一步证实了离体保存不灌洗犬肝的有效时限(25℃3小时,15℃4小时)。认为该时限能够满足临床完成肝移植的需要,并分析探讨了犬肝离体保存期间功能变化的特点。