Clusters of proliferating endothelial cells and smooth muscle cells in rabbit carotid arteries

Schwarz and Benditt found clustering of replicating cells in aortic endothelium in 1976 and discussed how homeostasis of the arterial wall is maintained through this nonrandom distribution of replicating cells. However, it is still unclear how cells of vascular walls turnover. In order to address this issue, we evaluated distribution of the cells in mitotic cycle, labeled by Ki67‐immunostaining, in serial histological sections of twelve carotid arteries of six adult male Japanese rabbits. As a result, a total of 1713 Ki67‐positive endothelial cells (ECs) and 1247 Ki67‐positive smooth muscle cells (SMCs) were identified. The Ki67‐positivity rate in ECs and SMCs were about 0.048% and 0.0027%, respectively. Many of the Ki67‐positive cells clustered in two (EC, 37%; SMC, 33%), three to four (EC, 8%; SMC, 28%), and five to eight cells (EC, 5%; SMC, 10%). Clusters having more than eight cells were not found. Thus, it can be speculated that the cell division of proliferating ECs and SMCs occur four times at most. These novel findings offer great insights for better understanding of the mechanism that underlies cell number regulation of the blood vessel.     

镁合金网联合兔颗粒骨植入在兔体内异位成骨的实验研究

目的:使用普通和带涂层的镁合金网联合自体颗粒骨植入大兔体内,观察颗粒骨的愈合情况和镁合金网的降解速度.方法:用微弧氧化法在镁合金网表面制成20μm的涂层.将金属网制成直径为8 mm,高度为16 mm的圆柱状.分为A、B、C三组,分别为明胶海绵、普通镁合金网和20μm涂层镁合金网制作圆筒.取新西兰大白兔的自体的髂骨,制成细小颗粒骨,填充于圆筒中,植入白兔的臀部肌袋中.分别在第4、8周测血清镁离子并给予X线检查,观察植入物形态并且骨荧光标记镜下检测.结果:涂层20μm的镁合金网降解速度要慢于普通镁合金网,在X线和直观观察下在3组颗粒骨的愈合过程中未见明显差异;3组大白兔在不同时间段的血清镁离子浓度比较,差异无统计学意义(P＞0.05);镜下观察荧光染色带分布和间隔的紧密程度依次是C组最致密、B组次之、A组较为稀疏;3组植入物在大白兔体内未见明显排异反应,组织相容性较好.结论:镁合金植入物有较好的组织相容性,镁合涂层可减慢降镁合金解速度,并且对于骨的生长具有一定的促进作用.具有一定的临床意义.     

兔眼球后Tenon囊下灌注Bevacizumab的眼内通透性研究

目的：比较兔眼球后Tenon囊下灌注和玻璃体腔注射bevacizumab后玻璃体和血清中的浓度,并观察bevacizumab视网膜荧光显影,探讨bevacizumab球后Tenon囊下灌注的眼内通透性和眼外给药途径的可行性。

方法：实验用健康成年新西兰兔20只,随机分为A组和B组,A组均单眼接受单次玻璃体腔注射1.25mg bevacizumab(1.25mg/0.05mL),B组均单眼单次Tenon囊下灌注5mg bevacizumab(5mg/0.2mL)。1、3d后抽取玻璃体和血液,使用双抗体夹心Elisa检测玻璃体和血清中bevacizumab药物浓度,比较两组中玻璃体和血清内bevacizumab浓度差异,并通过激光共聚焦观察视网膜免疫荧光。

结果：给药1d后,A组和B组玻璃体腔内bevacizumab药物浓度分别为254.40±13.65、1.60±0.32μg/mL。A组和B组血清内bevacizumab药物浓度分别为0.55±0.15、0.63±0.05μg/mL,两组血清bevacizumab浓度比较差异无统计学意义(t=1.168,P=0.277)。给药3d后,A组和B组玻璃体腔内bevacizumab药物浓度分别为236.80±8.70、1.40±0.23μg/mL,A组和B组血清内bevacizumab药物浓度分别为0.66±0.17、0.64±0.14μg/mL,两组血清内bevacizumab浓度比较差异无统计学意义(t=0.207,P=0.841),两种给药方式视网膜各层荧光分布均能明显显现。

结论：给药1、3d后玻璃体腔注药组在玻璃体腔内bevacizumab药物浓度要明显高于Tenon囊下灌注组,玻璃体腔内注射是较为有效的给药途径,而球后Tenon囊下灌注也能使bevacizumab进入玻璃体腔而且达到完全抑制VEGF活动所需的浓度(>500ng/mL),并能至少持续3d以上,两种给药方法在血清中均能检测到较高浓度的bevacizumab,且两者浓度差异无统计学意义(P>0.05),两种给药方式视网膜各层荧光分布均能明显显现,提示两种给药方式药物均能作用于视网膜各层。     

Non‐clinical safety evaluation of single and repeated intramuscular administrations of MAGE‐A3 Cancer Immunotherapeutic in rabbits and cynomolgus monkeys

The MAGE‐A3 recombinant protein combined with AS15 immunostimulant (MAGE‐A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non‐clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE‐A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE‐A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post‐injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3‐month treatment‐free period (2/5 per gender per group). Local and systemic reactions and MAGE‐A3‐specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post‐mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post‐injection), but returned to normal after 1–8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment‐related macroscopic findings were recorded after the treatment‐free period. MAGE‐A3‐specific antibody and T‐cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE‐A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys. Copyright © 2014 GlaxoSmithKline Vaccines. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.     

加味痛泻要方对家兔体外结肠平滑肌的作用

[目的]观察加味痛泻要方对家兔体外结肠平滑肌的作用。[方法]用生理记录仪记录正常及药物致痉孪肠管蠕动曲线,分别加入加味痛泻要方40、80、160 mg/ml,记录用药后兔结肠的蠕动曲线。分别计算给药前、后的抑制率和拮抗率。[结果]加味痛泻要方不仅对正常家兔结肠蠕动呈明显抑制作用,而且对平滑肌兴奋药,包括乙酰胆碱及氯化钡引起的平滑肌痉挛均有明显的对抗作用,具有剂量依赖性。[结论]加味痛泻要方可以很好地改善肠痉挛,对家兔体外结肠平滑肌的非规律性收缩有明显的缓解作用,推测该方可能是通过拮抗乙酰胆碱M受体起作用。     

Altered anomeric specificity of glucose-induced insulin release in rabbits with duct-ligated pancreas

Ligation of the pancreatic duct in rabbits provokes a decrease in the insulin and glucagon content of the pancreas, and may lead to chronic hyperglycemia. The insulin secretory behavior of the perfused pancreas is perturbed in duct-ligated animals, and this is illustrated in several respects:

Ultrastructural changes of venous autologous bypass grafts in rabbits: Correlation of patency and development

Summary Electron microscopic techniques were used to correlate the patency of venous autografts in rabbits with ultrastructural changes within a time range from 5 days to 6 months p.o. Early degenerative changes of the grafts include endothelial desquamation followed by fibrin deposition or platelet adhesion, mural edema and extensive medial and adventitial degeneration. The regeneration of the grafts is due to an early adventitial vascularization (10 days p.o.), which enables the surviving smooth muscle cells and fibrocytes to proliferate and to develop an arterial-like vessel. The organization of the 6-month-old graft is comparable to the carotid artery of the animal, but shows, in contrast, many features of a muscular artery.

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Zusammenfassung Mit Hilfe elektronenmikroskopischer Methoden wurde versucht, die Funktionsfähigkeit autologer Venentransplantate mit ultrastrukturellen Veränderungen in einem Zeitraum von 5 Tagen bis 6 Monaten p. o. zu korrelieren. Frühe degenerative Veränderungen der Transplantate umfassen Endothelabschilferungen mit anschließender Fibrin- bzw. Plättchenauflagerung, murale Ödeme und ausgedehnte mediale und adventitielle Degenerationen. Die Regeneration der Transplantate wird ermöglicht durch eine frühe adventitielle Kapillarisierung (10 Tage p. o.), die die überlebenden glatten Muskelzellen und Fibrozyten in die Lage versetzt, zu proliferieren und ein arterienähnliches Gefäß zu entwickeln. Der Aufbau der 6 Monate alten Vene ist vergleichbar mit der Arteria carotis communis der Tiere, zeigt aber im Gegensatz dazu überwiegend Merkmale einer Arterie vom muskulären Typ.

     

Utilization of oxygen by the contractile apparatus is disturbed during reperfusion of post-ischaemic myocardium

Post-ischaemic ventricular function remains depressed ( = myocardialstunning) despite nearly normal coronary blood flow during reperfusion.In order to illuminate the causes of this phenomenon, we studiedthe relationship between ventricular function and myocardialoxygen consumption (MVO_2tot) in experiments on 15 isolated rabbithearts perfused with erythrocyte suspension (hct=30%). Leftventricular systolic function was assessed by measuring aorticflow (ml. min ^–1), peak systolic pressure (L VP_max), dPldt_max,and early relaxation in terms of dPldt_min during control and30 min after the onset of reperfusion, following 20 min globalno-flow ischaemia. The pressure-volume area was calculated asa measure of total mechanical energy. The external mechanicalefficiency (E_ext) was assessed from stroke work and MVO_2torBothcontractile efficiency (E_con= inverse slope of the MVO₂-PVArelationship) and MVO₂ of the unloaded contracting heart (MVO_2unl=basal MVO₂ + MVO₂ for excitation-contraction coupling) werecalculated using pressure-volume area and MVO_2tot Results: At matched heart rate (149 ± 30 vs 147 ±31 min ^–1; mean ± SD) and end-diastolic volume(1.3 ± 0.2 ml), the systolic variables were significantlydecreased in the stunned myocardium: aortic flow: 38 ±13 vs9 ± 11 ml. min ^–1, LVP_max: 112 ±19vs 74±18mmHg, and dP/dt_max: 1475 ±400 vs 1075±275 mmHg. s^–1. Likewise, dP/dt_min was significantlyimpaired (– 1275 ±250 vs – 975 ±250).The decrease in pressure-volume area (570 ±280 vs 270±200mmHg.ml. 100g^–1) was not statistically significant. In contrast,both E_ext (0.75±0.29 vs 0.18±0.26 arbitrary units)and E_con (31 ± 18 vs 14± 7%) were significantlydecreased, whereas MVO_2tot (40±9 vs 34±8µl.beat^–1. 100g^–1) and MVO_2unl (26±9 vs22±6µl.beat^–1. 100g^–1) were not. Summary: Ventricular function after brief episodes of ischaemiais decreased whereas MVO_2tot is maintained, i.e. external efficiencyis decreased. MVO₂ for the unloaded contraction remained unchanged,indicating that MVO₂for excitation-contraction coupling is inappropriatelyhigh for the depressed contractile state. The decreased contractileefficiency indicates further that O₂ utilization of the contractileapparatus is disturbed during reperfusion.     

Hepatitis E virus isolated from rabbits is genetically heterogeneous but with very similar antigenicity to human HEV

Rabbit hepatitis E virus (HEV) in China may represent a novel HEV genotype, although no consensus has been reached. It is unclear whether the ORF2 capsid protein containing the immunodominant epitopes from rabbit HEV differs from those of human HEV. In this study, 661 bile samples collected from domestic rabbits in Jiangsu province, eastern China were amplified by RT‐nPCR using a set of HEV universal ORF2 primers. All 42 (6.4%) positive PCR products were sequenced. Phylogenetic analysis using the ORF2 sequences of 557 bp in length showed the Jiangsu isolates were separate from HEV genotypes 1, 2, 3, 4, avian HEV and rat HEV, and clustered together with rabbit HEV sequences. These 42 isolates were divided into five branches including two newly identified in the present study. Comparison with rabbit HEV sequences from China available in GenBank, using a 298 bp ORF2 segment, showed these sequences clustered together into a unique rabbit HEV clade, and were divided into eight sub‐branches with high genetic heterogeneity. In addition, 267 serum samples collected from domestic rabbits, serial serum samples from two rhesus monkeys experimentally infected with HEV genotype 1 or 4, and serial serum samples from two New‐Zealand rabbits infected experimentally with rabbit HEV were tested simultaneously by EIA using recombinant truncated ORF2 capsid proteins derived from rabbit and human HEV. The virtually identical results obtained suggest that rabbit and human HEV ORF2 antigens contain very similar immunodominant epitopes. All these data are helpful to identify the biological characteristics of the newly identified rabbit HEV. J. Med. Virol. 85:627–635, 2013. © 2013 Wiley Periodicals, Inc.