Non-surgical alternatives to invasive procedures in mice

We have developed and validated catheterization protocols in mice that allow for simultaneous infusion and sampling. A sampling catheter was inserted in the lateral vein of the tail, while the animals were infused either intravenously or intragastrically through a second catheter placed in the contralateral lateral vein or via an intragastric catheter, respectively. The applicability of these methods of infusion and blood sampling were validated by conducting urea kinetics utilizing stable isotopes. These non-surgical procedures are non-invasive, inexpensive, fast to perform and animals do not require a recovery period before their use.     

Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery

The goal of this study was to examine the efficacy of liver-targeted gene delivery by chitosan-DNA nanoparticles through retrograde intrabiliary infusion (RII). The transfection efficiency of chitosan-DNA nanoparticles, as compared with PEI-DNA nanoparticles or naked DNA, was evaluated in Wistar rats by infusion into the common bile duct, portal vein, or tail vein. Chitosan-DNA nanoparticles administrated through the portal vein or tail vein did not produce detectable luciferase expression. In contrast, rats that received chitosan-DNA nanoparticles showed more than 500 times higher luciferase expression in the liver 3 days after RII; and transgene expression levels decreased gradually over 14 days. Luciferase expression in the kidney, lung, spleen, and heart was negligible compared with that in the liver. RII of chitosan-DNA nanoparticles did not yield significant toxicity and damage to the liver and biliary tree as evidenced by liver function analysis and histopathological examination. Luciferase expression by RII of PEI-DNA nanoparticles was 17-fold lower than that of chitosan-DNA nanoparticles on day 3, but it increased slightly over time. These results suggest that RII is a promising routine to achieve liver-targeted gene delivery by non-viral nanoparticles; and both gene carrier characteristics and mode of administration significantly influence gene delivery efficiency.     

Rat liver-targeted naked plasmid DNA transfer by tail vein injection

High levels of foreign gene expression in mouse hepatocytes can be achieved by "hydrodynamics-based transfection," the rapid injection of a large volume of a naked deoxyribonucleic acid (DNA) solution into the tail vein. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice and, thus, are more suitable for some biomedical research. Recently, we demonstrated that hydrodynamics-based transfection can also be used to deliver naked plasmid DNA into the normal rat, which is more than 10 times larger than the mouse. We performed the tail vein injection using a syringe with a winged needle equipped with an external tube. Injection of a lac Z expression plasmid, pCAGGS-lac Z by this technique resulted in the exclusive detection of beta-galactosidase in the liver. We also injected a rat erythropoietin (Epo) expression plasmid, pCAGGS-Epo (800 microg). Maximal Epo gene expression was achieved when a 25-mL injection volume (approx 100 mL/kg body wt) was transferred within 15 s.     

Non-surgical cannulation of the vena cava for chronic blood collection in mature swine.

A nonsurgical cannulation technique for blood collections from mature swine was evaluated. Primiparous Yorkshire-Landrace sows (n = 6) received an indwelling jugular vein cannulae for 7 days duration. Recannulation was performed at monthly intervals for a total of 14 months. During cannulation, sows were restrained in a standing position using a rope snout snare. A 12-gauge by 10 cm needle was inserted into the jugular vein. Sterilized polyvinyl chloride tubing was advanced through the needle into the vein and a blunted 18-gauge needle and attached intermittent injection hub was inserted into the free end of the tubing. Surgical tape was used to form a butterfly on the tubing by suturing the tape to the animals' skin. Foam padding, livestock cement, and elastic tape helped to keep the tubing in position. Problems with cannulae patency and maintenance were few. No behavioral problems or systemic signs of illness were noted and necropsy examinations performed after the final cannulation revealed few abnormalities associated with chronic intermittent cannulation. This technique provides a safe, quick, effective means for multiple and repeated cannulae placement for blood collection from mature swine with minimal effects on the animal and without the risks associated with surgical techniques.     

Continuous intravenous infusion in athymic (nude) rats: an animal model for evaluating the efficacy of anti-cancer agents

The athymic (nude) rat (rnu/rnu) has been used for a number of years in research into various human tumours involving xenotransplantation. We now report the validation of a continuous intravenous infusion method in nude rats using a tail cuff tether, which enables the study of the efficacy of novel anti-cancer materials in this mutant strain, using intravenous infusion and with no restriction of the animals or of the tumour implantation sites by jackets. Ten animals each had a cannula surgically implanted into the vena cava via the femoral vein and exteriorized via a tail cuff. Animals were housed singly in conventional cages following surgery. Following a recovery period of 5 days all animals were continuously infused with physiological saline at an infusion rate of 0.5 ml/h for a further 37 days. Body weights and food consumption were recorded weekly. Blood samples were taken approximately 14 days post-surgery and analysed for haematology and clinical chemistry parameters. All animals were successfully cannulated, and no unexpected adverse clinical signs were noted during the recovery period and the 37 days of infusion. The results demonstrate that it is possible to surgically cannulate the femoral vein of athymic (nude) rats and infuse them in conventional cages for a period of up to 37 days with minimal adverse effects. The minimal restraint required provides benefits both to the animal and to the conduct of studies such as assessment of tumour growth in the absence of a jacket. Recent work has demonstrated that the same techniques can be successfully applied to the nude mouse.     

Technique for long-term infusion into the inferior mesenteric artery of unrestrained rats

A technique for long-term infusion into the inferior mesenteric artery was developed which allows simple and reliable regional infusion into the colorectal segment of unrestrained rats. The cannulation system consists of an injection port 'In Stoppers' as a flow swivel, connected to an injection needle, which is inserted into a polyethylene tube protected by a steel spiral. During infusion the animals are free to move in the cage with access to food and water ad libitum. The method is suitable for regional chemotherapy as well as for studies of colorectal tumours in rats. In this study 73% of the cannulae remained functional for continuous infusion over a 15 day period.     

Electroshock raises pentylenetetrazol threshold: Possible role of inosine

A series of 15 single electroshocks administered through electrodes applied to the scalp of mice raised the threshold to pentylenetetrazol-induced seizures as determined by a tail vein infusion method. The same stimulus increased brain inosine and hypoxanthine. Phenytoin, which blocks the increase in inosine produced by this stimulus, reversed the elevation in threshold by electroshock. The effect of electroshock on threshold may be mediated by inosine.     

In vivo BUdR labeling of mammalian chromosomes

A technique is described for labeling mammalian chromosomes in vivo with BUdR. Rats and mice are given BUdR by tail vein infusion over a 24-h period at a concentration of 25 μg/g wt/h. Metaphase cells that have gone through two or three cycles of DNA synthesis reveal characteristic differential chromatid fluorescence after staining with Hoechst dye. Sister chromatid exchanges can then be easily detected in these cells.     

Trichinella spiralis: vascular recirculation and organ retention of newborn larvae in rats

The recirculation of Trichinella spiralis newborn larvae was studied in inbred AO rats. Newborn larvae collected after in vitro incubation of adult T. spiralis worms for 2 or 24 hr were injected into rats through the tail vein or hepatic portal vein. Blood samples from the femoral vein, hepatic portal vein, and abdominal aorta were collected at intervals from 1 min to 24 hr after larval injection. Newborn larvae of both ages (24 hr or 2 hr old) persisted in femoral vein blood for less than or equal to 5 hr after injection, but they could be detected in portal vein blood by 24 hr after injection. The injection of larvae into a tail vein or the portal vein did not influence the pattern of larval circulation, although there was a 1-5 min delay in newborn larval appearance time after injection into the portal vein. Transcapillary migration through tissue and back to the circulation was evident in the appearance of newborn larvae in the thoracic duct lymph up to 24 (occasionally 48) hr after tail vein injection of newborn larvae. During the course of a natural primary infection, no evidence for trapping of larvae in the mesenteric lymph node could be found despite direct larval migration through this organ. Injected newborn larvae were retained in the lungs, and small numbers could be recovered 24 hr after intravenous injection. We conclude that a proportion of newborn larvae recirculates within the vasculature for several hours; a smaller population extravasates but can reenter the circulatory system via the lymphatics. Furthermore, some newborn larvae are found in organs rich in capillaries up to 24 hr after their entry into the blood.     

The Tip of the Tail Needle Affects the Rate of DNA Delivery by Bacteriophage P22

The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host’s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a “knob” with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host.     

EGFP标记的A20细胞构建小鼠B细胞播散型淋巴瘤模型

目的:A20细胞是来源于同系Balb/c小鼠大B细胞淋巴瘤的细胞系,能通过尾静脉接种建立小鼠B细胞播散型淋巴瘤的模型,但由于该细胞系缺乏细胞表面特异性标志而难于监测。本研究使用增强型绿色荧光蛋白(EGFP)标记A20细胞,试图建立易于监测的小鼠B细胞播散型淋巴瘤模型。方法:用含增强型绿色荧光蛋白(EGFP)的慢病毒载体将标记基因EGFP转入A20细胞,通过流式分选出EGFP+的A20细胞,体外培养后通过尾静脉注射接种于同系Balb/c小鼠体内,用流式细胞仪监测其外周血EGFP+细胞的百分率。当小鼠出现消瘦、毛发竖立、嗜睡等体征时,将小鼠处以安乐死;取动物脏器行石蜡包埋、病理切片、HE染色。结果:尾静脉注射1×106细胞于6只Balb/c小鼠体内,接种后15天可在外周血中检测到EGFP+细胞,平均生存时间为29.6±0.8天;在肝脏、脾脏、脊椎和淋巴结等多脏器成瘤,流式检测瘤细胞EGFP表达阳性。结论:经尾静脉注射接种A20细胞可建立小鼠B细胞播散型淋巴瘤模型,A20细胞经含EGFP的慢病毒标记后易于通过流式进行监测,为通过动物体内试验评价靶向治疗的疗效提供了保证。     

树[鼠句]的基本实验操作方法

目的对树[鼠句]抓取和保定、采血、灌胃基本实验技术方法进行探讨,逐步规范树[鼠句]实验技术。方法选用成年树[鼠句]进行抓取和保定,分徒于法和器具法（自制捕捉保定袋）,对130只树[鼠句]采用尾静脉、股动（静）脉两种采血方法;采取人用8号胃管可对树[鼠句]绎口灌胃给药。结果所采用徒手、器具的方法抓取和保定树[鼠句]均能有效地控制动物,不会发生动物死亡或很少逃逸;两种方法都顺利采集到所需血量,股动（静）脉单次最大采血量可达2mL而不损伤动物;12只树[鼠句]连续灌胃10d,成功率100％。结论自制的捕捉保定袋经济实用,摸索的几种树[鼠句]实验技术方法具有操作简便、安全、快捷等优点。     

Domain organization and polarity of tail needle GP26 in the portal vertex structure of bacteriophage P22

The attachment of tailed bacteriophages to the host cell wall as well as the penetration and injection of the viral genome into the host is mediated by the virion tail complex. In phage P22, a member of the Podoviridae family that infects Salmonella enterica, the tail contains an approximately 220 A elongated protein needle, previously identified as tail accessory factor gp26. Together with tail factors gp4 and gp10, gp26 is critical to close the portal protein channel and retain the viral DNA inside the capsid. By virtue of its topology and position in the virion, the tail needle gp26 is thought to function as a penetrating device to perforate the Salmonella cell wall. Here, we define the domain organization of gp26, characterize the structural determinants for its stability, and define the polarity of the gp26 assembly into the phage portal vertex structure. We have found that the N-terminal 27 residues of gp26 form a functional domain that, although not required for gp26 trimerization and overall stability, is essential for the correct attachment to gp10, which is thought to plug the portal vertex structure. The region downstream of domain I, domain II, folds into helical core, which exhibits four trimerization octad repeats with consensus Ile-xx-Leu-xxx-Val/Tyr. We demonstrate that in vitro, domain II represents the main self-assembling, highly stable trimerization core of gp26, which retains a folded conformation both in an anhydrous environment and in the presence of 10% SDS. The C terminus of gp26, immediately downstream of domain II, contains a beta-sheet-rich region, domain III, and a short coiled coil, domain IV, which, although not required for gp26 trimerization, enhance its thermodynamic stability. We propose that domains III and IV of the tail needle form the tip utilized by the phage to penetrate the host cell wall.     

Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the 'hydrodynamics-based procedure.' The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene in the rat liver by injecting fCAGGS-Epo, an Epo-expressing PCR-amplified DNA fragment, via the tail vein. After injection of 5 pmol fCAGGS-Epo (10 microg) or pCAGGS-Epo (18.4 microg), plasmid DNA, the serum Epo levels peaked at week 1, then persisted for at least 12 weeks. Transgene-derived Epo secretion resulted in significant erythropoiesis. These results demonstrated that transfer of PCR-amplified DNA fragments into the rat liver via rapid tail vein injection can be achieved. This method may provide a useful means for studying the physiologic function of a putative gene.     

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane‐labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.     

脐带间充质干细胞移植对慢性试验性肝损伤的治疗作用

目的观察脐带间充质干细胞（UC-MSC）对慢性实验性肝损伤的治疗作用并探讨其分子生物学机理。方法 50只7周龄的NOD/SCID小鼠注射四氯化碳（CCL4）制备慢性肝损伤模型后,应用随机数字表的方法随机将实验小鼠随机分成2组：模型组（25只）和UC-MSC移植组（25只）。UC-MSC移植组通过尾静脉注射移植1×106 UC-MSC,模型组注射同样体积的PBS。分别于移植后1、2、3和4周收集肝组织,应用免疫组织化学,RT-PCR和Western blot的方法分析细胞移植前后肝组织的病理生理学特征的变化。采用t检验和方差分析进行统计学分析。结果 UC-MSC移植治疗后肝组织表达人肝细胞特异性AFP,Alb,和内皮细胞特异性CD31,Flk-1。细胞移植4周后v WF标记的血管密度明显增加,同时伴有部分的肝功能改善,谷丙转氨酶（ALT）水平从（55.71±11.33）U/L减至（36.75±12.80）U/L（P〈0.05）。此外,本研究结果表明UC-MSC分泌几种重要的生长因子HGF,FGF-2,VEGF,和VEGF受体通过旁分泌的途径发挥肝组织修复的功能。结论在CCL4诱导的慢性肝损伤模型肝组织,人UC-MSC可以分化成肝细胞样细胞和内皮细胞样细胞,同时旁分泌多种细胞生长因子修复损伤的肝细胞,并伴有肝功能的改善。认为UCMSC移植或许成为将来肝脏损伤疾病一个重要的治疗选择。     

Evaluation of different routes of administration and biodistribution of human amnion epithelial cells in mice

Placenta is a non-controversial and promising source of cells for the treatment of several liver diseases. We previously reported that transplanted human amnion epithelial cells (hAECs) differentiate into hepatocyte-like cells, resulting in correction of mouse models of metabolic liver disease or acute hepatic failure. As part of preclinical safety studies, we investigated the distribution of hAECs using two routes of administration to efficiently deliver hAECs to the liver. Optical imaging is commonly used because it can provide fast, high-throughput, whole-body imaging, thus DiR-labeled hAECs were injected into immunodeficient mice, via the spleen or the tail vein. The cell distribution was monitored using an in vivo imaging system over the next 24 h. After splenic injection, the DiR signal was detected in liver and spleen at 1, 3 and 24 h post-transplant. The distribution was confirmed by analysis of human DNA content at 24 h post-transplant and human-specific cytokeratin 8/18 staining. Tail vein infusion resulted in cell engraftment mainly in the lungs, with minimal detection in the liver. Delivery of cells to the portal vein, via the spleen, resulted in efficient delivery of hAECs to the liver, with minimal, off-target distribution to lungs or other organs.     

Long-term blood-sampling technique in piglets

A technique to enable long-term blood sampling from piglets aged 2-3 months is described. Piglets were housed individually in expandable cages and a heparinized polyurethane catheter was inserted into the external jugular vein. A technique was used which prevented the catheter from pulling out of the vein with growth of the animals. Blood samples could be obtained for more than 1 month, and levels of cortisol, glucose, white blood cell count, haematocrit, rectal temperature and heart rate were compared for samples obtained from simulated conventional venopunctures and from the cannula using this technique. It was shown that restraint and needle pricks raised these levels considerably.     

A rapid and non-surgical procedure for jugular catheterization of pigs

A rapid and non-surgical method for jugular catheterization in pigs was set up in 30 piglets of 6.2 kg, 23 pigs of 46 kg and 84 kg and two lactating multiparous sows. The animal was restrained on a V-shaped table (piglets) or with a rope around the mandible (slaughter pigs and sows). The vein was located with the Vacutainer system and a wire guide was inserted into the Vacutainer needle up to the vein lumen. When the needle was removed, the catheter was inserted over the wire guide and advanced until it penetrated the skin and thereafter, the vein wall. The catheter was fixed outside by a large tape and coiled inside a patch just behind the ears. The technique utilizes readily available material and is no more risky for the animal than a single blood sampling. Moreover, it can be performed within 15 to 20 min (including animal restraint) within pens. This new approach might have important implications not only for research purposes by facilitating repeated blood samplings but also for projects which require a rapid and easy method for testing of any kind of pharmaceutical or other type of products under husbandry conditions.     

右美托咪啶对脓毒症大鼠肾脏Toll受体4 的影响

目的：观察右美托咪啶对脓毒症大鼠肾脏功能及Toll 受体4 表达的影响,探讨减轻脓毒症损害的潜在治疗方案。方法：选择 雄性SD 大鼠作为研究对象,采取盲肠结扎穿孔方法建立脓毒症模型;取模型制备成功的大鼠按照数字随机法分为3 组,A 组为 空白对照组,泵注1.0 mL/kg 生理盐水,B 组为对照组,术毕泵注1.0 mL/kg 生理盐水,维持10 min,持续泵注NS 1.0 mL/kg·h,C 组为实验组,术毕给予右美托咪定5 滋g/kg,维持10 min,持续泵注右美托咪定4.5 滋g/kg·h,3 组均持续泵注24 h,并于6 h、12 h、 24 h各组分别取8 只大鼠检测中性粒细胞明胶酶相关脂质运载蛋白（NGAL）表达水平进行比较,并观察比较3 组研究对象尿素 氮（BUN）、肌酐清除率（Ccr)以及肾脏组织Toll 受体4水平及TLR4 mRNA水平。结果：C 组大鼠应用右美托咪定后,BUN于6 h、 12 h、24 h分别为（13.4± 1.2）mmol/L、（12.5± 1.3）mmol/L、（11.4± 1.1）mmol/L,均低于A 组、B组,均P<0.05;Ccr 于6 h、12 h、24 h 分别为（77.4± 9.9）mL/min、（75.4± 10.2）mL/min、（78.5± 11.3）mL/min,均明显低于A 组、B 组,P<0.05;C 组6 h、12 h、24 h NGAL 表达水平分别为（39.2± 9.9）pg/mL、（41.6± 9.8）pg/mL、（38.2± 9.9）pg/mL,均明显低于A组、B组,P<0.05;C 组泵注6 h、12 h、24 h Toll受体4 水平及TLR4 mRNA 水平均低于A 组、B 组,P<0.05。结论：右美托咪啶可降低脓毒症大鼠Toll 受体4表达,有助于减 轻脓毒症炎性反应与减轻肾损害,具有重要临床价值。