Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei

The objective of this study was to determine fertilization rates and developmental ability of porcine oocytes following injection of round spermatid and round spermatid nucleus with artificial activation either 2 h before or immediately after injection. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization compared with that following injection without stimulation or with stimulation immediately after injection. Incidences of formation of 2 pronuclei and of apposition were not different in oocytes following intracytoplasmic spermatid and spermatid nucleus injection. Chromosome analysis revealed that most oocytes were diploid either following round spermatid or round spermatid nucleus injection. There was no diploid set of chromatin in oocytes at 20 h following sham injection. At 6 d following injection blastocoele formation was seen in the oocytes following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of the oocytes developed to the blastocyst stage 6 d following sham injection. The average cell numbers of blastocysts 8 d after injection of spermatid and spermatid nucleus were 99 and 87, respectively. These results suggest that electrical stimulation before injection enhances the incidence of fertilization following round spermatid injection in the pig. Our study also indicates that either the round spermatid or it's nucleus can be used to produce viable embryos by injection into unfertilized porcine oocytes.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

三种药物对大鼠糖尿病性白内障延缓效应的比较

目的验证腹腔注射葡萄糖能否成功建立大鼠糖性白内障模型,并且探讨黄芩苷点眼、牛磺酸点眼、牛磺酸腹腔注射以及Vc腹腔注射对大鼠糖性白内障的延缓效应及短期治疗作用。方法按照雄鼠每次2.5mL,雌鼠2 mL剂量注射30%葡萄糖生理盐水建立大鼠糖性白内障模型,给药同期进行,点眼每日2次,腹腔注射每日1次。从注射葡萄糖第3天开始对各组晶状体进行裂隙灯检查,并定期测定体重、饮食量等相关指标。结果实验结果表明葡萄糖腹腔注射第3天即可使大鼠晶状体出现空泡现象。各组药物的延缓效应与短期治疗效应相同,依次为牛磺酸注射、黄芩苷点眼、牛磺酸点眼以及Vc注射。结论 30%葡萄糖腹腔注射能快速建立大鼠糖性白内障模型,且比半乳糖注射建模成本低,成模率高。4%牛磺酸腹腔注射以及0.1%黄芩苷在预防和治疗大鼠糖性白内障方面效果显著。     

注水式结肠镜检查法的研究进展

注水式结肠镜检查法是指用注水代替传统的注气,通过向肠腔内注水以扩张肠管,循腔进镜的肠镜检查方法。自1984年《新英格兰杂志》首次报道应用传统的注气式结肠镜检查失败时,应用辅助注水的方式帮助寻找肠腔走行,有利于内镜顺利通过憩室段以来,经过三十年的发展与研究,注水式结肠镜检查法较传统的注气式结肠镜检查具有减轻患者腹痛、缩短进镜时间及提高盲肠插管成功率等方面的优势。本文主要就注水式结肠镜检查法的历史起源、发展历程、研究进展、存在的问题以及应用前景等进行了综述。     

Effects of an N-methyl-d-aspartate receptor agonist and its antagonist CPP on the levels of dopamine and serotonin metabolites in rat striatum collected in vivo by using a brain dialysis technique

3-((±)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is an antagonist at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. In the present study, levels of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) were measured after intracerebroventricular injection of NMDA, CPP or both in rat striatum using a brain dialysis method. The injection of NMDA produced a significant increase in DOPAC level. HVA level was also increased by NMDA injection. The level of 5-HIAA was not affected by NMDA injection. The injection of CPP had no effect on DOPAC, HVA and 5-HIAA levels. The injection of CPP restrained the increase of DOPAC and HVA levels induced by NMDA injection. The results suggest that intracerebral injection of NMDA may increase dopamine release from rat striatum, but have no effect on serotonin release. Furthermore, CPP inhibits NMDA induced release of dopamine.     

小鼠睾丸注射不同转染试剂和注射方法对外源基因表达的影响

为探讨不同转染试剂(LipofectamineTM LTXPLUSTM、Lipofectamine2000和纳米化聚酰胺-胺型树枝状聚合物(PAMAM-D))和睾丸注射方法 (睾丸网注射、曲精细管注射和间质注射)对转基因小鼠生产效率的影响,将pEGFP-C1质粒分别与不同转染试剂混合后,按照不同的注射方法注入小鼠睾丸内,30 d后检测小鼠精子密度、活力、精子阳性率以及配种后仔鼠转基因阳性率。结果 3种转染试剂对小鼠繁殖性能影响由小到大依次为LipofectamineTM LTXPLUSTM、Lipofectamine 2000和PAMAM-D。转染后LipofectamineTM LTXPLUSTM、Lipofectamine 2000和PAMAM-D组精子的GFP阳性率分别为35.65%±0.69%、12.86%±0.35%和10.04%±0.20%。配种后仔鼠的PCR阳性率分别为29.17%、13.70%和5.88%。3种不同注射方法对小鼠睾丸都造成损伤,由小到大依次为睾丸网注射、曲精细管注射和睾丸间质注射,三者的阳性精子比例分别为35.13%±1.727%、15.13%±1.457%和0%,配种后仔鼠的PCR阳性率分别为33.3%、12.5%和0%。结果表明,LipofectamineTM LTXPLUSTM和睾丸网注射对小鼠睾丸的损伤最小,并能获得较高的转染效率。     

基于多角度成像光谱仪(MISR)数据的中国野火烟羽喷射高度

烟羽喷射高度是烟气羽流运动一个关键驱动因素,决定了烟气气溶胶在大气中的寿命、顺风运输扩散路径及其对大气环境的影响程度.本研究对最新的多角度成像光谱仪(MISR)野火烟羽高度数据库中的原始数据进行提取和处理,获取中国区域的野火羽流高度相关参数,采用统计分析的方法研究野火烟羽喷射高度的变化情况,探究了火灾特征(燃烧生物质类...     

氯通道在葛根素注射液致溶血反应中的作用

目的：观察阻断和激活氯通道对葛根素注射液致家兔溶血反应的影响,探讨氯通道在葛根素注射液溶血反应中的作用。方法：将不同浓度的葛根素注射液（0.75、1.5、3、6、12 mg/ml）与家兔红细胞悬液共孵育6 h,使用细胞图像记录系统观测葛根素注射液是否诱导红细胞溶血,酶标仪、流式细胞仪检测红细胞溶血率,并观测激活和阻断氯通道对葛根素注射液溶血作用的影响。结果：葛根素注射液可引起家兔红细胞体外溶血,在所观察的1.5 mg/ml~12 mg/ml范围内,溶血效应呈浓度依赖性增强（n=3,P<0.01）。氯通道阻断剂Tamoxifen （20 μmol/L）、ATP （10 mmol/L）可有效抑制葛根素注射液的溶血作用（n=3~5,P<0.01）;而使用低浓度ATP （50 μmol/L）激活氯通道,则显著增强葛根素注射液的溶血作用（n=4,P<0.01）。结论：葛根素注射液的体外溶血效应呈浓度依赖性,氯通道激活与葛根素注射液诱导的溶血反应密切相关。     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

Antibiotics in surgical treatment of acute abscesses.

A four-way, double-blind, prospective trial of treatment of abscesses by incision, curettage, and primary closure with and without antibiotic cover (clindamycin injection before operation or capsules after operation, or both) was conducted. There was no appreciable difference in mean healing time between the patients given both the antibiotic injection and the antibiotic capsules and those given the injection and placebo capsules, whereas healing times in those given the placebo injection and antibiotic capsules or placebo only were appreciably longer. Four of the patients who were not given the antibiotic injection developed bacteraemia; one patient who was given the antibiotic injection also developed a bacteraemia, but this was caused by clindamycin-resistant bacteria. These results show that a single injection of an effective antibiotic before operation is sufficient to protect the patient against bacteraemia and permit optimum healing.     

Reversible Inhibition of Acetylcholine Synthesis and Behavioural Effects Caused by 3-Bromopyruvate

3-Bromopyruvate inhibits pyruvate decarboxylase in brain homogenates and causes a 90% drop in acetylcholine tissue content at a concentration of 2 mM. Stereotaxic injection of 3-bromopyruvate into the basal forebrain causes after 7 days a 40% drop of acetylcholine concentration and pyruvate decarboxylase activity in the cortex and hippocampus, and greater decreases at the site of injection. However, values return to normal 18 days after injection. Choline acetyltransferase is partially inhibited only at the site of injection after 7 days. Choline transport and choline concentration are not affected at either 7 or 18 days after injection. Impairments in spontaneous alternation and in retention of passive avoidance were seen only 7 days after the injection. The results suggest that stereotaxic injection of bromopyruvate can induce discrete reversible cholinergic lesions on a time scale useful for behavioural experiments and for comparison with neurodegeneration.     

Molecular dynamics simulation of formation and growth of CdS nanoparticles

Monodispersed semiconducting nanoparticles are usually synthesised in a liquid medium using injection of an appropriate solution. A key factor in attaining a narrow particle size distribution (PSD) is the temporal separation of the nucleation and growth stages, where the former takes place during the injection. Faster injection produces a larger number of nuclei and a narrower PSD. The injection speed is expected to affect the diffusion of the ions in the solution and to create uniformly high supersaturation for a short period of time. In this paper, we study the growth of CdS nanoparticles during the injection by molecular dynamics simulation. A solution of Cd ions is injected into the simulation cell that contains sulphure ions; the variation of the PSD and its mean and variance are studied as functions of the injection velocity. Higher injection velocities produce narrower PSDs and smaller particles, hence providing a precise method for controlling both.     

Minimizing the pain of local anesthesia

We studied the effect of depth of lidocaine injection into the skin, rate of injection, and temperature of the solution on pain experienced. The intervals of onset and duration of anesthesia were also evaluated. Intracutaneous instillation of lidocaine at body temperature (37 degrees C) is no less painful than injection at room temperature (21 degrees C), but superficial wheal-producing dermal injection is uniformly much more painful than that into the deep dermal-subcutaneous tissue region. Rapid injection almost always hurts more than slow. Full anesthesia to pinprick is produced immediately with superficial injection and is present 5 to 6 minutes after deep injection. We suggest that the best method for minimizing the discomfort of inducing local anesthesia is to use a syringe fitted with a No. 30 needle and to inject the smallest amount necessary slowly into the deep dermal-subcutaneous tissue as the needle is being slowly withdrawn.     

Inhibition of renin release following left circumflex bradykinin injection in dogs

We examined the renin secretory response to bradykinin (BK) injection into the left circumflex coronary artery (LCx) in dogs. Studies were conducted in anesthetized, carotid sinus denervated dogs which had been maintained on a low sodium diet. A 25 ga needle was inserted into the LCx for injection of BK (0.15 micrograms/kg). The rate of renin secretion (RS) was obtained during a 30 min control period, at 5 min after a non-hypotensive hemorrhage (10 ml/kg), at 1, 3 and 5 min after BK injection and at 15 min after the reinfusion of withdrawn blood. Four series of studies were conducted. Series I: BK injection into the LCx, Series II: saline injection into the LCx (sham), Series III: intravenous injection of BK, and Series IV: BK injection into the LCx in dogs with prior renal denervation. RS was suppressed by 80% (P less than 0.05) 5 min after injection of BK into the LCx. Saline injection (sham) into the LCx or intravenous BK administration did not inhibit RS. Furthermore, suppression of RS was not present in dogs with prior renal denervation. These results indicate that BK injection into the LCx causes a prompt reduction in the rate of RS and that this response is reflexively mediated by the renal nerves.     

In vivo effect of epidermal growth factor,interleukin-1beta,and interleukin-1RA on equine preovulatory follicles

Paracrine factors have significant effects during folliculogenesis. Because of various morphological features, the mare is a convenient model to study in vivo the effects of factors involved in periovulatory events. In the present work, epidermal growth factor (EGF; experiment 1, n = 49 mares) and interleukin-1beta and interleukin-1RA (IL-1beta and IL-1RA, respectively; experiment 2, n = 80 mares) were injected intrafollicularly to evaluate the influence of these factors on in vivo maturation of equine preovulatory follicles. A transvaginal ultrasound-guided injection was performed when the diameter of the dominant follicle reached 30-34 mm. In experiment 1, the four experimental groups were 1) EGF group, intrafollicular (i.f.) injection of EGF (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum; 2) control group, no injection; 3) PBS group, i.f. injection of 2 ml of PBS plus i.v. injection of physiological serum; 4) crude equine gonadotropins (CEG) group, i.f. injection of PBS plus i.v. injection of CEG (20 mg). In experiment 2, groups 3 and 4 were the same as in experiment 1, but groups 1 and 2 were changed as follows: 1) IL-1beta group, i.f. injection of IL-1beta (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum; 2) IL-1RA group, i.f. injection of IL-1RA (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum. In each experiment, cumulus-oocyte complexes from dominant/injected follicles were collected by transvaginal ultrasound-guided aspiration 38 h after intrafollicular injection. Cumulus morphology and oocyte nuclear stage were assessed. Additionally, in experiment 2, 40 mares were used to determine the time of ovulation after treatments. Our results indicate that intrafollicular injection of EGF or PBS induced lower cumulus expansion and oocyte maturation rates compared with the CEG group (P < 0.05). In experiment 2, the IL-1beta and CEG groups showed the same expansion rate, the same oocyte maturation rate, and the same ovulation distribution. On the other hand, the intrafollicular injection of IL-1RA, as PBS, did not induce follicle and cumulus-oocyte complex (COC) maturation. In conclusion, we confirmed that the technique of intrafollicular injection can be used in the mare to study the role of specific molecules. We demonstrated for the first time in mares that the injection of EGF did not influence in vivo COC maturation. In contrast, IL-1beta injection into the dominant follicle induced in vivo oocyte maturation and the ovulation process whereas IL-1RA seemed to block these mechanisms.     

Low-volume jet injection for intradermal immunization in rabbits

Background

This study tested a low-volume (20–30 μl/20–30 μg DNA) jet injection method for intradermal delivery of a DNA vaccine. Jet injection offers the advantages of a needle-less system, low-cost, rapid preparation of the injected DNA solution, and a simple delivery system. More than one construct can be injected simultaneously and the method may be combined with adjuvants.

Results

Low-volume jet injection targeted delivery of a DNA solution exclusively to the dermis and epidermis of rabbits. A three injection series of plasmid DNA, encoding the Hepatitis B Surface Antigen stimulated a humoral immune response in 2/5 rabbits. One rabbit developed a significant rise in antibody titer after 1 injection and one following 2 injections. There were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10–100 times higher than jet injection.

Conclusion

Low-volume jet injection is a simple, effective methodology for intradermal DNA immunization.     

Gene transfer by biolistic process

Gene transfer into somatic tissues is a tool for both the study of gene function in the basic science laboratory and for gene therapy and genetic immunization in the clinic. Biolistic processes can be used to deliver both viral and nonviral vectors into somatic tissues. This review discusses the advantages and disadvantages of three biolistic processes: jet injection, microparticle bombardment, and needle and syringe injection. Jet injection and needle and syringe injection can be used to deliver both viral and nonviral vectors. Both jet injection and microparticle bombardment can be used to target a broad range of tissues. Needle and syringe injection has been most widely used in muscle tissue. The choice of which biolistic process to use is dependent on the specific application.     

Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization.

In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.     

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.     

Tolerance to morphine in the rat: its prevention by naloxone.

Development of tolerance after a single injection of morphine in the Wistar-Lewis rat can be estimated by the attenuation of the response to a second injection of morphine given three days later. If naloxone is given 35 minutes after the first morphine injection and after the appearance of measurable analgesia, attenuation of the effects of the second morphine injection is not seen. It appears that naloxone blocks the development of tolerance to morphine even if given after the morphine-receptor interaction responsible for analgesia has been initiated. The temporal relationship between the prior injection of morphine and the subsequent administration of naloxone is being explored.