Intravitreous injection for establishing ocular diseases model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a Hamilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given. To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45 degrees injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.     

Implications of experimental technique for analysis and interpretation of data from animal experiments: outliers and increased variability resulting from failure of intraperitoneal injection procedures

Intraperitoneal (i.p.) injections are widely used in laboratory animal experiments. This technique has a failure rate that is typically reported to be of the order of 10-20%. It is not apparent that failures of i.p. injection and their consequences for the experimental results are as widely recognized as the use of the technique. We illustrate the consequences of i.p. injection failure for the analysis and interpretation of several bioassays. We suggest approaches to data analysis that should be considered, and emphasize the need to recognize and allow for the possibility of i.p. injection failure in the analysis and interpretation of laboratory animal experiments involving this technique.     

Vector design for optimal protein expression

Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is essential for stability, as well as for translation efficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of the sequences [5'UTR, 3'UTR, and poly(A) signal] are suggested.     

Une approache pratique des résistances psychologiques aux traitements par injections intracaverneuses (I.I.C.)

Intra-cavernous injection of vasoactive substances has dramatically modified the medical approach of erectile dysfunction. There are psychological and ethical difficulties to this therapeutic procedure, and it is likely that they do contribute to the high drop out rate encountered with this method. According to our clinical experience, we suggest several attitudes in order to facilitate patient's and partner's acceptance:

? There is no opposition between psychotherapy and intra-cavernous injection, they may be regarded as complementary.

? It is advisable to find means to reduce the fear induced by self injection, by reassuring the patient, and making the act easier by choosing the most simple injection material. The pharmacological action must be explained, as well as the side effects.

? Pharmacologically induced erection is often described as artificial, although intra-cavernous injection of vasoactive substances is a symptomatic treatment just as many other symptomatic treatments. It compensates a functional insufficiency, restoring a physiological state very close to normal as far as erection is concerned, but has no effect on sexual excitation, glans intumescence and the development of the ejaculatory climax reflex. The sexuel relation's erotization lays only within the couple, and remains entirely apart from any iatrogenic effect.

? Should the treatment remain secret? Inta-cavernous injection must not systematically be performed withtout the partner knowing about it, although the act should remain discrete and intimate.

One should not encounter psychic or ethical difficulties in erectile dysfunction treatment with intra-cavernous injection of vasoactive substances, provided that it has been properly prescribed, well explained, and that it is part of a psychotherapy. It is doubtless that many refusals or therapy withdrawals are caused by poor or lack of technical initiation, on one hand, and by not taking into account the psychic and ethical aspects of the method, on the other hand.     

Varicose veins; a practical approach to treatment

Adequate treatment of varicose veins requires thorough mapping of perforating veins, communicating veins and "blow out" areas. Combined ligations, stripping and injection of sclerotic substances after operation is the most effective regimen of therapy. The technique of stripping is facilitated by isolating the saphenous vein at the ankle, inserting the stripper from below upward, then making a transverse groin incision over the palpable stripper. The tip of the stripper should be twice the diameter of the vein to be removed. Stripping should be done with the patient in the Trendelenburg position. All patients must be examined at regular intervals after operation and injection of sclerosing material carried out as necessary.     

Cluster of postinjection abscesses related to corticosteroid injections and use of benzalkonium chloride

Benzalkonium chloride (BC) is an unreliable disinfectant. A matched case-control study and environmental investigation were conducted to determine the cause of and risk factors for a cluster of postinjection abscesses at a private medical clinic where BC was used as a disinfectant. Twenty-eight case-patients who had an abscess at the injection site were matched with 126 control patients who had received an intramuscular injection at the clinic on the same day. Risk factors for abscess development in a multivariable logistic model were corticosteroid injection and being female. All case-patients had received a corticosteroid injection from a multidose vial. Cultures of abscesses from 20 of 23 case-patients grew Pseudomonas aeruginosa. Cultures of BC prepared at the clinic also grew P aeruginosa, suggesting that BC was the source of infection. Injection site cleaning with BC did not appear to be the route of infection since use of BC at the time of injection was not associated with abscess development. A more likely route of infection was injection of contaminated corticosteroid from multidose vials that could have been inoculated with pseudomonads via needle puncture after vial septa were wiped with contaminated BC. Benzalkonium chloride should not be used to clean injection vial septa or injection sites.     

VARICOSE VEINS—A Practical Approach to Treatment

Adequate treatment of varicose veins requires thorough mapping of perforating veins, communicating veins and “blow out” areas. Combined ligations, stripping and injection of sclerotic substances after operation is the most effective regimen of therapy.The technique of stripping is facilitated by isolating the saphenous vein at the ankle, inserting the stripper from below upward, then making a transverse groin incision over the palpable stripper. The tip of the stripper should be twice the diameter of the vein to be removed. Stripping should be done with the patient in the Trendelenburg position.All patients must be examined at regular intervals after operation and injection of sclerosing material carried out as necessary.     

Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid

We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.     

An approach to the focal presentation of chemical stimuli

An olfactometer is described that presents temporally-discretepulses of stimuli to individual chemosensory structures. Thedevice is based on standard pressure injection techniques inwhich pulses of compressed air eject nanoliter volumes of upto six stimulants from small diameter glass micropipettes. Thedevice should be readily adaptable to different chemoreceptorpreparations.     

Human insulin

The two human insulins of clinical importance are (a) semisynthetic human insulin prepared from pork pancreas by enzymatically substituting threonine for alanine-the last amino acid in the beta chain-thereby transforming pork insulin in vitro to human insulin; and (b) biosynthetic human insulin synthesized biotechnologically in Escherichia coli-K12. Using this latter technique, it is possible to produce mass quantities of highly purified insulin for the treatment of insulin-dependent diabetics, avoiding the problems inherent in supplies of insulin produced from animal pancreas. It has been suggested that to avoid confusion the two human insulins should be called semisynthetic human insulin of pork origin and biosynthetic human insulin of E. coli origin, respectively. These insulins have four advantages over highly purified animal insulins: (a) they induce lower titers of circulating insulin antibodies; (b) their subcutaneous injection is associated with fewer skin reactions; (c) they are absorbed more rapidly from the injection site; and (d) less degradation occurs at the site of injection. These data indicate that newly diagnosed insulin-dependent diabetes, particularly in children, should be treated with either of the two human insulins. The warranty against inadequate supplies of insulin offered by biosynthetic human insulin makes the use of pork insulins unnecessary and beef insulins totally useless.     

Adrenocorticotropic hormone-releasing activities of interleukins in a homologous in vivo system

We compared adrenocorticotropin-releasing activities of several interleukins in a homologous or heterologous in vivo system. Intravenous injection of rat interleukin-1 alpha significantly increased plasma adrenocorticotropin in conscious, freely-moving rats 30 min after the injection, and the effect was 10 times greater than that of human interleukin-1 alpha. Rat interleukin-2 affected plasma adrenocorticotropin in a much slower manner and increased its levels significantly 120 min after the injection. Human interleukin-2 had no effect on plasma adrenocorticotropin. Thus, species difference in the experimental system should be considered to assess the physiological significance of cytokines in the neuroendocrine system.     

The effect of agitation system, temperature of the spray liquid, nematode concentration, and air injection on the viability of Heterorhabditis bacteriophora

Entomopathogenic nematodes (EPNs) are a group of potentially effective bio-insecticides to which Heterorhabditis bacteriophora, an active ingredient of several commercialised products, belongs. The application of the spray liquid with a motorised hydraulic sprayer introduces several stress factors to EPNs that may reduce their viability. Therefore, the effects of the agitation system, initial temperature of the spray liquid, EPN concentration, and additional air injection on the viability of EPNs were studied. The results clearly illustrate that the hydraulic agitation caused significantly more reduction in viability than the mechanical agitation. A lower temperature of the initial spray liquid, however, yielded a significantly higher EPN viability compared to a higher temperature after hydraulic mixing and so did air injection while EPN concentration did not significantly influence viability. Thus, the results clearly suggest that, with hydraulic agitation, both the re-circulation stress and the temperature increase significantly decreased the EPN viability, while air injection significantly improved it. Therefore, specific application conditions of living organisms in sprayer design and during application should be considered. Furthermore, spray water of less than 20°C should be used to keep temperature under control.     

Susceptibility of volume stress in Colisa fasciatus.

Intraperitoneal injections of 0.6% NaC1, commonly used as injection vehicle, at 10 mul/g, but not at 5 mul/g. elicit significant (p less than .05) transitory erythropenia, leucopenia, and thrombopenia at selected post-injection time intervals in female Colisa fasciatus, a fresh water teleost. It is suggested that administration of large volumes of injection fluid per gram body weight of experimental animals should be avoided lest it might distort physiological parameters, particularly those relating to blood components, under study.     

Disseminated intravascular coagulation following intravenous Corynebacterium parvum injection in the rat

Summary Corynebacterium parvum (C. parvum) was administered by a single IV injection (14 mg/kg) to rats, and platelet counts, plasma fibrinogen concentrations and thrombin clotting times were monitored for up to 7 weeks. During this time histological and ultrastructural studies were also conducted. Thrombocytopoenia, hypofibrinogenaemia, and prolongation of the thrombin clotting time rapidly followed C. parvum injection and were accompanied by the appearance of platelet clumps and fibrin within blood vessels in a variety of tissues. This initial episode of disseminated intravascular coagulation (DIC) subsided 12–24 h after injection, but a more prolonged second episode of DIC occurred 1–3 days after injection. The results suggest that caution should be observed when systemic immunotherapy with C. parvum is proposed.     

THE STATHMOKINETIC METHOD IN VIVO TIME-RESPONSE WITH SPECIAL REFERENCE TO CIRCADIAN VARIATIONS IN EPIDERMAL CELL PROLIFERATION IN THE HAIRLESS MOUSE

Groups of hairless mice were injected i.p. with a stathmokinetic dose of 0·15 mg colcemid at seven different times of the day and animals killed 0, 15 and 30 min, 1, 2, 3 and 4 hr after the injection. The proportion of cells in metaphase and ana/telophase was determined in histological sections. The results showed a transient accumulation of metaphases about 30 min after the injection, followed by an increase in metaphases from 1 to 4 hr. Therefore, no value before 1 hr after the colcemid injection should be used in calculations of the mitotic rate. The presence of circadian rhythms with high mitotic activity in the morning and low activity in the evening was confirmed. It is shown by regression analyses that the accumulation period of 4 hr is sufficiently short to reflect circadian variations in epidermal cell proliferation and that the 4-hr accumulation value alone is sufficient to estimate the mitotic rate.     

Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level

The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation.     

High-resolution intracellular recordings using a real-time computational model of the electrode

Intracellular recordings of neuronal membrane potential are a central tool in neurophysiology. In many situations, especially in vivo, the traditional limitation of such recordings is the high electrode resistance and capacitance, which may cause significant measurement errors during current injection. We introduce a computer-aided technique, Active Electrode Compensation (AEC), based on a digital model of the electrode interfaced in real time with the electrophysiological setup. The characteristics of this model are first estimated using white noise current injection. The electrode and membrane contribution are digitally separated, and the recording is then made by online subtraction of the electrode contribution. Tests performed in vitro and in vivo demonstrate that AEC enables high-frequency recordings in demanding conditions, such as injection of conductance noise in dynamic-clamp mode, not feasible with a single high-resistance electrode until now. AEC should be particularly useful to characterize fast neuronal phenomena intracellularly in vivo.     

Treatment of acute agitation in psychotic disorders

Several psychotic disorders, including schizophrenia, may be associated with symptoms of acute agitation and aggression. While drug treatment of agitation is often essential, non-pharmacological interventions, both environmental and behavioral, also play important roles in the complex management of agitated patients. The most extensively used psychotropic drugs are parenteral formulas of conventional antipsychotics and benzodiazepines. Recently, injection forms of two second generation antipsychotics, olanzapine and ziprasidone, have become available. Both drugs have shown adequate efficacy and tolerability in several double-blind trials of intramuscular administration in acutely agitated psychotic patients. Compared to conventional medication, injection forms of the new antipsychotics may have a faster onset of action and more favorable profile of adverse events. Alternative approaches to injection administration include liquid drug formula, orally disintegrating tablets and wafers, treatment initiation with high doses, or rapid dose escalation. Evidence suggests that second-generation antipsychotics should be among the first-line choices in the treatment of agitation in acute psychosis.     

Severe Depressive Mood Changes Following Slow-release Intramuscular Fluphenazine Injection

Sixteen patients in whom schizophrenia was initially diagnosed and who were treated with fluphenazine enanthate or decanoate developed severe depression for a short period after the injection. In five cases this depression is thought to have been responsible for suicide. In 8 out of 10 cases the depression responded to electroplexy (E.C.T.). It is recommended that patients who are treated with fluphenazine should be carefully supervised.