Age-dependent decline of blood-brain barrier P-glycoprotein expression in the canine brain

The efflux transporter P-glycoprotein serves as a major molecular gatekeeper at the blood-brain barrier. It has been suggested that a reduction of P-glycoprotein activity with aging might enhance exposure of brain tissue to exogenous and endogenous compounds thereby contributing to the development of neurodegenerative diseases.Brain tissue from owner-kept dogs renders an excellent tool to study the impact of aging on the background of variable environmental and genetic influencing factors. Therefore, we determined expression rates of P-glycoprotein in canine post-mortem tissue from 23 non-laboratory dogs. P-glycoprotein expression in the parahippocampal cortex exhibited a negative correlation with age. Analysis of the area labeled for P-glycoprotein in dogs aged >100 months revealed a 72% drop in P-glycoprotein expression as compared to young adults aged 23-36 months. Respective data from the dentate hilus and dentate gyrus indicated an earlier drop with a reduction by 77 and 80% in dogs aged 37-99 months in comparison with younger individuals. In contrast to the decline observed with aging in dogs without plaques, P-glycoprotein expression rates rather tended to increase with further aging in dogs with plaque formation.In conclusion, the thorough analysis of P-glycoprotein expression rates in non-laboratory dogs revealed a significant decline with aging. The data strongly support the concept that age-dependent changes might predispose to neurodegenerative diseases. In the early pathogenesis of Alzheimer's disease which is modelled by diffuse plaques in the canine brain, an up-regulation of P-glycoprotein might act as a compensatory mechanism to enhance Abeta efflux from the brain. Future studies are necessary to further evaluate the correlation between Abeta deposits and P-glycoprotein expression in different phases of the disease.     

Serial blood cultures from canine stored whole blood held at room temperature for 24 h

To determine if 30- to 35-day-old units of canine stored whole blood held at room temperature over 24 h will reveal bacterial contamination when exposed to repeated sampling. The design of the study is prospective, controlled. Ten units of stored whole blood from ten client owned volunteer blood donor dogs were used. A sterile blood bag spike was inserted aseptically as a port for repeated sampling for culture into ten separate units of 30- to 35-day-old canine stored whole blood held at room temperature for 24 h. Aerobic and anaerobic bacterial culture samples were collected every 4 h over 24 h. No bacterial growth was detected in any sample at any time point from the ten bags used. Canine stored whole blood, collected and stored for 30 to 35 days according to accepted transfusion guidelines, can be aseptically spiked and held at room temperature for 24 h without evidence of bacterial contamination or proliferation.

T-cell-derived cytokines,nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune

It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n = 6) and Leishmune^® (n = 6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune^®, with the Leishvaccine triggering a mixed, IFN-γ and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune^® induced an immunological pattern characterized by enhanced levels of IFN-γ, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune^®, the ability of both immunobiologicals to activate T-cell-derived IFN-γ synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune^®) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.     

Effect of bovine cumulus–oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes

Purpose

To investigate the ability of medium conditioned with bovine cumulus–oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries.

Methods

Cumulus–oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO₂, 5% O₂ and 90% N₂. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO₂, 98% air.

Results

The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate.

Conclusions

The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.     

Transboundary Spread of Brucella canis through Import of Infected Dogs,the Netherlands,November 2016–December 2018

Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notified to the authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis–endemic areas, posing a threat to the naive autochthonous dog population and humans.     

Antibacterial Effect of Antibiotic Solution on Cellular Viability in Canine Veins

Pretreatment of tissue by using antibiotics is a critical step to prevent microbial contamination before venous transplantation. In this study, the optimal time and temperature of antibiotic solution treatment for maintaining cellular viability with antibacterial effect were investigated. The antibiotic-nutrient solutions were composed of cefoxitin, lincomycin, vancomycin, and polymyxin B in RPMI-1640 medium. After various antibiotic solution treatment times (4, 8, and 12 h) and temperatures (4, 25, and 37 degrees C), the viabilities of cells dissociated from veins (jugular vein, femoral vein, superior vena cava, and inferior vena cava) were determined. Double staining by Griffonia simplicifolia agglutins-fluorescein isothiocyanate (GS1-FITC) and propidium iodide was used. To measure the antibacterial effect of the antibiotic solution, canine veins were artificially infected by 3 kinds of bacteria (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae) and were treated by antibiotic solutions as viability test conditions. After the treatment with the antibiotic solution, the tissue was minced, and the homogenized tissue fraction was cultured on standard method agar. The colony that seemed to be resistant to the antibiotic solution was counted. At 37 and 25 degrees C, the viability of whole cells decreased significantly Asymptotic Significance 2-tailed (Asymp.Sig 2-tailed) < 0.05 after 4 h of antibiotic solution treatment, whereas at 4 degrees C it began to reduce significantly after 8 h of treatment. By antibiotic solution treatment at all 3 temperatures for 4 h, no significant difference in viability of the endothelial cells and whole cells was observed. To maintain the donor vein's cellular viability until transplantation, antibiotic solution treatment for 4 h at 4 degrees C is assumed to be appropriate.     

复合生物陶瓷在尖牙再移植中的应用

人体尖牙在颌面、牙弓中占重要位置,对维持人颌面的正常形态、功能起重要作用。尖牙异位埋伏阻生、严重错位、移位,影响人体面容美观。本文采用拔除异位尖牙,并移植回尖牙应在牙列位置,用复合生物陶瓷一人工骨充填固位的方法治疗11例,经一年半观察,取得良好效果。     

犬膀胱移行上皮细胞的体外连续培养及生物学特征观察

目的探讨犬膀胱移行上皮细胞体外连续培养和纯化方法,为进一步构建组织工程尿路上皮组织提供实验依据。方法无菌获取幼犬膀胱组织,0.125%胰蛋白酶消化获取单细胞悬液,于上皮细胞无血清培养基中培养。采用0.05%胰蛋白酶和0.02?TA消化、纯化细胞,动态观察细胞形态变化和增殖情况,MTT法绘制生长曲线;透射电镜观察细胞超微结构;SABC法对培养的细胞进行免疫组织化学鉴定;并采用流式细胞仪检测不同代次细胞的细胞周期和倍体水平。结果采用酶消化法能从4～6cm2的膀胱黏膜组织中分离获取(1～5)×106个细胞。原代培养接种24h后可见大量细胞贴壁,第7天细胞生长融合达80%～90%,细胞排列呈典型的铺路石样。MTT法测定第3代细胞在传代培养7d生长达高峰;传代并纯化的细胞纯度高,无成纤维细胞污染,至第4～6代细胞仍保持良好形态。透射电镜下,可见上皮细胞特征性张力微丝和细胞间桥粒连接。细胞角蛋白AE1/AE3免疫组织化学染色,胞浆呈棕黄色阳性反应。不同代次细胞均为二倍体细胞。结论酶消化法能从较少的膀胱组织中分离获取足量、较纯的膀胱移行上皮细胞,细胞在无血清培养基中能连续传代扩增,可以满足进一步构建组织工程尿路上皮组织的需要。     

Heterogeneity in epithelium-dependent responses

The airway epithelium exerts a profound influence on the responsiveness of bronchial smooth muscle to both contracting and relaxing agents. This may be due to the release of an epithelium-derived factor or factors. There is a considerable heterogeneity in the effects of the epithelium between orders of bronchi, between species, and between pharmacologic agents. Such heterogeneity may reflect variations in the release and/or effect of the epithelium-derived relaxing factor(s). This report demonstrates that: (1) there is a basal and a stimulated release of the factor, (2) the prominence of different types of release varies between species, (3) the effect of the epithelium on relaxation of bronchial smooth muscle is greatest in the presence of high degrees of cholinergic tone, (4) the effects of the epithelium are not mediated via cyclic GMP, and (5) the epithelium-derived relaxing factor is not nitric oxide.     

犬囊状动脉瘤微弹簧圈栓塞治疗前后的超声研究

目的探讨囊状动脉瘤模型的声像图和血流动力学特征以及超声对其血管内栓塞效果的评价。方法利用用显微外科技术于犬颈总动脉制成三种类似于人类的囊状动脉瘤模型，其中侧壁型12例、分叉型4例、末端型6例。术后7～14d彩超观察瘤腔、载瘤动脉及瘤体内血流动力学改变。栓塞后再行彩超检查观察其疗效，并与MRA、DSA及病理检查结果对比。结果三种类型动脉瘤均有相似的血流模式，瘤腔内血流分为三个血流区：1)沿远侧瘤壁进入的流人区；2)沿近侧瘤壁出去的流出区和3)中心缓慢涡流区。流人区峰速高于流出区。②侧壁型动脉瘤内血流速较慢，易形成部分性血栓，而分叉型和末端型动脉瘤内血流速高。③血管内治疗后，血流显示19例动脉瘤腔闭塞，2例瘤颈部有残腔，1例弹簧圈略脱出瘤颈导致载瘤动脉狭窄。④超声结果与MRA、DSA及病理结果完全一致。结论①各型动脉瘤模型有相似的血流模式。②侧壁型动脉瘤顶部易形成部分血栓。③双功超声能观察载瘤动脉及瘤体内血流动力学改变，可作为动脉瘤及栓塞治疗效果无创评价的方法之一。