India Ink-Gelatin Vascular Injection of Skeletal Tissues

Dogs under sodium pentothal anesthesia were given 100 mg of heparin intravenously and, 15 min later, exsanguinated. A mixture consisting of Higgins India ink #4415, 1 part, and a 14% solution of calfskin gelatin, 1 part, was injected (at 37° C) into the femoral artery of a disarticulated hindlimb. A pump-type 5 ml hypodermic syringe was connected with a gravity-feed reservoir that contained the injection mass, with a filter in the outflow from the reservoir. Veins severed by the disarticulation of the limb permitted a free flow of the injection fluid out of the vascular tree. After injection of about 400 ml of fluid by pumping the syringe, the limb was chilled (—20° C) to solidify the gelatin. Specimens from cleaned bones were sawed out and fixed in Zenker-formalin solution for 24 hr, then decalcified with 6% sulfosalicylic acid. The technique was completed by embedding in celloidin and sectioning. Blood vessels in both cortical and trabecular regions were completely filled by the injection mass.     

嗜酸乳杆菌耗尽培养上清液对小鼠在体肠管运动的影响

目的研究嗜酸乳杆菌LA14菌株耗尽培养上清液(spent culture supernatant,SCS)对小鼠在体肠管运动的影响。方法昆明种小鼠50只,随机分为5组。各组分别灌胃给予SCS、活菌菌液、SCS+活菌菌液25ml/(kg.次)、硫酸阿托品注射液10 mg/(kg.次)和等容积生理盐水,每天2次,连续3 d。末次给药后1 h,各组灌胃给予印度墨汁0.2 ml/只,20 min后处死,测定小肠全长及墨汁在小肠内推进距离,计算墨汁推进率和抑制率。另取小鼠60只,随机分为6组。各组分别灌胃给予SCS、活菌菌液、SCS+活菌菌液25 ml/(kg.次)、思密达散剂1.5 g/(kg.次)或等容积生理盐水,每天2次,连续3 d。末次给药后1 h,除生理盐水组外,其余各组灌胃给予甲硫酸新斯的明注射液2 mg/kg,10 min后各组灌胃给予印度墨汁0.2 ml/只,10 min后处死,同上述方法进行测定与计算。结果在SCS组、SCS+活菌组及阿托品组,正常小鼠的墨汁推进率明显降低,与生理盐水组比较差异有非常显著性(P0.01)。给予新斯的明造模后,小鼠的肠蠕动亢进,墨汁推进率明显升高,与生理盐水组比较差异有非常显著性(P0.01),而给予SCS、活菌、SCS+活菌以及思密达后,由新斯的明所造成的小鼠小肠运动亢进受到明显抑制,小鼠墨汁推进率显著下降,与模型组比较差异有非常显著性(P0.01)。结论嗜酸乳杆菌SCS能显著抑制正常小鼠的小肠推进运动;同时SCS及活菌能明显拮抗新斯的明所致的小鼠小肠运动亢进。     

Injection of Lymphatics: With Colored Cedar Oil; With Plastic

(1) The oil mass consists of: cedar oil, 1; color in oil (a paint pigment, e.g., Prussian blue), 1; and toluene, 2, parts by volume. To use, add 1 ml of diethyl ether to each 10 ml of mass, mix thoroughly and inject into the fresh organ with a very fine glass or metallic needle. Heat the organ in water at 50-60° C before starting the injection, massage gently after injection, then fix. For macroscopic studies, fix 5 days in 5% formalin, and dissect. For microscopic studies, fix at least 5 days in: formalin, 10 ml; Al₂(SO₄)₃, 2 gm; ZnSO₄, 2 gm; acetic acid, 4 ml; and distilled water, 90 ml. Dehydrate with dioxane, embed in paraffin and section at 10-20 μ. Stain with hematoxylin-eosin or with one of the following modifications of Van Gieson's formula: 1. 1% acid fuchsin, 10; picric acid (sat. aq.), 50; and 5% ZnSO₄, 40 volumes. 2. 1% acid fuchsin, 20; picric acid (sat. aq.), 80; and 5% CoSO₄, 40 volumes.

(2) The plastic mass consists of a 5-10% solution of Rhodopas (a vinyl copolymer) in acetone. Injection is made as with the oil mass except that a plastic squeeze-bottle and glass needle is preferable to a syringe. Indirect injection is used for both procedures, i.e., into the organ substance; not into a cannulated lymphatic vessel. After the plastic has hardened (24 hr), the unfixed tissue is subjected to corrosion by 5-10% NaOH in water.     

Granular biogenic amine-containing cells of the rat endometrium as components of the system of mononuclear phagocytes

To 10 non-inbred white mature rats 24 h before sacrifice 7.0 ml of Indian ink colloid solution has been injected intraperitoneally. Histochemical reactions of Falk-Hillarp (catecholamines, serotonin) have been performed on nonfixed cryostat slices of the uterus, those of Cross, Even, Rost (histamine) against non-specific esterase and acid phosphatase. Before carrying out these reactions, the slices have been examined under luminescent microscope LUMAM-I3 by means of luminescent and usual illumination in order to reveal cells, possessing autoluminiscence and containing phagocyted Indian ink particles. Presence of autoluminescence, phagocyted material, catecholamines and serotonin, histamine, nonspecific esterase, acid phosphotase, prostaglandin E2 in the same granular cells of the rat endometrium have been stated. All cellular properties revealed are specific for macrophages. A conclusion is made that granular biogenic amine-containing cells of endometrium can be considered as belonging to the system of mononuclear phagocytes.     

Ultrasound-guided intrafollicular treatment in mares

A technique for intrafollicular treatment with a transvaginal ultrasound-guided injection needle was developed using equine chorionic gonadotropin (eCG) as the test substance. An injection was made into one growing follicle of a wave when the follicles were 20 to 23 mm. The treated follicles were injected with 1000 iu of eCG in 0.2 ml saline solution and control follicles were injected with 0.2 ml of the saline vehicle (10 mares per group, 1 follicle per mare). The injection system used an inner 25-gauge needle and an outer 20-gauge needle inserted together through the needle-guide channel of a linear-array trans vaginal transducer. The outer needle was pushed through the vaginal wall and the inner needle was then advanced into the follicle during monitoring on the ultrasound screen. The turbulence in the follicular fluid associated with injection was observable on the screen. Seven follicles were successfully injected in each group. The follicular fluid in the control follicles remained anechoic until the follicle was no longer identifiable or ovulated. All 7 follicles in the eCG group showed ultrasonic indications of luteinization, based on the formation of an echogenic, thickened wall or area. Five of the 7 developed a central area that had the ultrasonic appearance of a blood clot similar to the appearance of a corpus hemorrhagicum. Ovulation was not detected in any of the eCG-treated follicles. The maximum post-treatment diameter of follicles was greater (P < 0.05) for the eCG group (32.7 +/- 3.8 mm) than for the control group (23.4 +/- 1.8 mm). The mean diameter for the first 5 days post-treatment (before the occurrence of an ovulation in any mare) was also greater (P < 0.002) in the eCG group (21.6 +/- 0.8 mm vs 19.6 +/- 0.8 mm). Results indicated that this novel research approach is practical and has potential for studies on folliculogenesis. The technique provides a research model between the extremes of an in vitro culture system and treatment of the whole animal.     

Distribution of ricin within the mammalian para-aortic lymph node. II. Comparison of the localization, after intramuscular dosage of colloidal gold-labelled ricin in vivo, with in vitro binding characteristics of the native toxin

Previous work has shown that, following an intramuscular injection of ricin, the toxin becomes localized within histiocytes in the sinuses of lymph nodes draining the 'wound' site. When ricin labelled with colloidal gold was similarly injected, it was found within the same lymphoid cells as seen with native ricin. Biologically inert Indian ink apparently follows a similar fate, as demonstrated by the appearance of carbon particles within sinus histiocytes, as soon as 1 h after intramuscular injection. When the binding in vitro of Indian ink or ricin toxin to sections of lymph node was examined, ricin was seen to bind to the surfaces of the same sinusoidal cells and also, with a much lower frequency, to follicular lymphocytes, whereas Indian ink failed to bind. This indicated an interaction between ricin and cell membrane components. Moreover, this binding was inhibited markedly by the galactose-containing disaccharide, lactose, a target sugar specified by the lectin binding site of ricin and to a much lesser extent by the monosaccharide mannose.     

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.     

Induced inflammatory process in the Antarctic fish Notothenia neglecta

We studied the ability of the Antarctic fish Notothenia neglecta to conduct an induced inflammatory process at 0°C. Indian ink was injected and a cotton suture thread was implanted into muscle of different groups of fish. After 1–2 days of Indian ink injection, the ink was diffused in the perimysium and there was hemorrhage and cellular infiltrate composed mainly of macrophages A (with few and small lysosomes) and neutrophils; after 7–15 days, there were macrophages A and some macrophages B (cytoplasm clear, lamellar cytoplasmic system forming interdigitations); after 30 days, there was Indian ink in the interior of macrophages A. The suture thread process takes place in two phases: the first (up to 7 days) with predominance of macrophages A and few neutrophils, and the second (15–30 days) with predominance of macrophages B. It can be concluded that N. neglecta is responsive to irritant stimulus with inflammatory process indicating adaptation to the antarctic environment. Received: 22 July 1997 / Accepted: 27 April 1998     

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.     

Comparison of Suprachoroidal Drug Delivery with Subconjunctival and Intravitreal Routes Using Noninvasive Fluorophotometry

Purpose

To determine whether exposure of sodium fluorescein (NaF) to the choroid-retina region in the posterior segment of the eye is greater with suprachoroidal injection when compared to intravitreal and transscleral routes.

Methods

Suprachoroidal injection, a new approach for drug delivery to the posterior segment of the eye was validated using a 34 G needle and Indian ink injections in Sprague Dawley rats, followed by histology. Delivery of NaF was compared in Sprague Dawley rats after suprachoroidal, posterior subconjunctival, or intravitreal injections. NaF levels were monitored noninvasively up to 6 hours using Fluorotron Master™, an ocular fluorophotometer Pharmacokinetic parameters were estimated using WinNonlin.

Results

Histological analysis indicated localization of India ink to the suprachoroidal space below sclera, following injection. NaF delivery to choroid-retina was in the order: suprachoroidal > intravitreal >posterior subconjunctival injection. Peak NaF concentration (C_max) in choroid-retina was 36-fold (p = 0.001) and 25-fold (p = 0.001) higher after suprachoroidal (2744±1111 ng/ml) injection when compared to posterior subconjunctival (76±6 ng/ml) and intravitreal (108±39 ng/ml) injections, respectively. NaF exposure (AUC_0–360min) to choroid-retina after suprachoroidal injection was 6-fold (p = 0.001) and 2-fold (p = 0.03) higher than posterior subconjunctival and intravitreal injections, respectively. Choroid-retina T_max was observed immediately after dosing with suprachoroidal injections and at 10 and 27.5 minutes, respectively, with subconjunctival and intravitreal injections.

Conclusions

Suprachoroidal injections are feasible in a rat model. Suprachoroidal injections resulted in the highest bioavailability, that is, the extent and rate of delivery of NaF to choroid-retina, when compared to intravitreal and posterior subconjunctival injections. Ocular fluorophotometry is useful for noninvasive monitoring of NaF in rats following administration by various routes including suprachoroidal route.     

Administration of semisynthetic human insulin by a spray injector

The use of Jet injection in insulin administration pointed out the question whether this route could affect insulin absorption and plasma insulin profiles. To compare plasma insulin profiles following an administration of an identical insulin dose by jet injection or by conventional subcutaneous route (syringe with needle) 8 healthy subjects (age 24-28 yrs., non obese) were given at 09.00 h of two different days 200 mU/kg/BW of human semisynthetic regular insulin (Novo Actarapid) alternatively subcutaneously by a syringe with needle or transcutaneously by jet injection (DG 77 - Sicim - Gorizia). Before insulin administration and then 15, 30, 60, 90, 120 and 180 minutes after, blood samples were drawn for plasma insulin and C-peptide determination. Higher plasma insulin levels after administration by jet were found at 15' and 30' minutes (62,58 +/- 6,31 v.s. 36,94 +/- 3,31 microunits/ml at 15' and 76,51 +/- 9,60 v.s. 51,65 +/- 9,95 at 30', p less than 0,01 and p less than 0,005, paired Student t test). No difference could be observed for the other times. C-peptide was found to fall to undetectable values, confirming the nearly total suppression of endogenous insulin production. It is concluded that total regular insulin absorption does not differ after transcutaneous jet injection or administration by syringe with needle, but in the first case it is faster. This last finding should be considered in planning insulin treatment schedules.     

Hereditary variability of plants under the action of exogenous DNA

Summary Injection of exogenous barley donor DNA into grains of barley recipient plants at the milk maturity stage, with a specially designed syringe, led to the appearance of transformed plants. The transformation (in rare cases) was caused by the unsheared DNA since the DNA passing through the syringe needle remained relatively stable (10⁶ to 10⁷ daltons) as was confirmed by DNA sedimentation analysis.14 plants grown from seeds injected with highly polymeric DNA containing close to 30 per cent protein had transformed pollen grains. In the 2nd generation only 2 plants from the 8 studied preserved these changes. In the progeny of these two plants, i.e., in the 3rd seed generation after injection, 82.1 per cent of plants preserved the transformed characters. The next, 4th generation, preserved a transformed phenotype in 89.6 per cent of plants.It was also shown that reversion to a recipient-like state was not always constant. We found the reversion of transformed properties (i.e., normal starch and two-rowed spikes) in 40 per cent of the 4th generation descendants of one of the plants which had lost the phenotypical expression of these properties in the 3rd generation but had them in the 2nd generation.The study of the morphological properties of transformed plants showed that with respect to phenotypic expression some characters were changed towards the donor type, some remained as in the recipients and some were of the intermediate type.     

Technical delivery of myogenic cells through an endocardial injection catheter for myocardial cell implantation

BACKGROUND: The next clinical frontier in the therapeutics of ischemic heart disease may involve the development and delivery of specific molecules and cells into the myocardium. The aim of the present study was to evaluate the efficiency and safety of the MyoStar injection catheter (Biosense-Webster Inc.) that has recently been developed to deliver molecules and cells to the myocardium. The 8 Fr (110 cm length) catheter comprises a navigation sensor with a 27 gauge needle at the distal tip. METHODS: Mouse myogenic cells (C2) were delivered to a tissue culture dish through different modalities: a standard laboratory pipette, a syringe needle (27 gauge) and the injection catheter. The cells were counted and monitored for growth and differentiation in the tissue culture immediately after delivery and two, three and six days later. Cells that were injected through a regular syringe needle or through the injection catheter demonstrated the same capacity to proliferate in tissue culture up to six days. RESULTS: The behavior of the cells in culture (fusion) was identical for the cells delivered to the tissue culture by a pipette or by the injection catheter. CONCLUSION: The results of the present study indicate that delivery of cells through the MyoStar injection catheter is a method with no significant loss or adverse effects to the cells along the path of the catheter. The catheter, which possesses both injection and navigation capabilities, can be used to deliver cell therapy to patients with ischemic heart disease.     

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.     

Effect of sucrose supplementation by stem injection on the development of soybean plants

Over the past half decade several stem injection methods have been developed for cereal plants. These methods allow researchers to administer solutions to cereal plants to study their effects on plant physiology. However, little work has been done to extend this technique to non-cereals An experiment was conducted to test an injection technique that could be suitable for soybean plants (Glycine max [L.] Merr,), and to study the effect of long-term injection of sucrose on the growth of soybean plants. An injection set-up comprising a supporting stand and a fluid injection system was established. Pressure was applied to the plunger of a 5 ml syringe using ceramic bricks to force test solutions into the plants. Solutions of 0, 150, and 300 g sucrose l^-1 were injected into soybean plants for 8 weeks starting at the seedling VC stage. Distilled water had the greatest uptake rate, followed by the 150, and then the 300 g sucrose l^-1 solutions. The overall average uptake during the injection period was 77.3 ml. Average sucrose uptake values were 11.8 and 13.5 g per plant for the 150 and 300 g sucrose l^-1 treatments. This represented approximately 65% of the total dry weight of the plants. Sucrose injection increased leaf area and pod number relative to the control plants. Nodule numbers were lower for sucrose injected treatments, but their dry weights were higher than the control. Above-soil dry matter was higher for the plants injected with 300 g sucrose l^-1 than those injected with water. The injection system tested was able to administer concentrated solutions into soybean plants for most of their period of growth and development. The sucrose supplementation had positive effects on soybean growth but suppressed photosynthesis.Key words: Soybean, sucrose, injection.      

Gas-powered jet injection compared with conventional methods of injection using lignocaine and technetium-99m.

Plasma concentrations of lignocaine and the clearance rates of technetium-99m were compared in 10 healthy volunteers after injection with a gas-powered jet system and a needle and syringe. The dosage was highly accurate with jet injection. Bioavailability was comparable for both methods.     

Uptake of India ink particles and latex beads by corneal fibroblasts

Summary The fate of India ink particles and polystyrene latex beads injected into the corneal stroma of rabbits was studied by the naked eye, light microscopy, and electron microscopy. All the injected ink particles or latex beads were unchanged in shape, size, and number for at least 6 months. India ink particles and latex beads were endocytosed by the corneal fibroblasts within 3–4 days after injection. Numerous ink particles were packed into vacuoles, 0.5–10 m in diameter, which occupy a large volume of the cytoplasm of the cell body and processes of fibroblasts in and near the injected area. Each latex bead, 0.72 m in diameter, is usually enclosed in one vesicle, and a large number of vesicles are distributed throughout the cytoplasm. In corneal tissue removed 10 min after injection of India ink and cultured for 3 or 7 days, uptake of many ink particles by the fibroblasts was seen. By this experiment, the contribution of the blood-derived cells was completely excluded, and it is more distinctly shown that the corneal fibroblast has a strong endocytotic activity.The uptake and long-term storage of ink particles and latex beads by the corneal fibroblast are reactions that protect the organ without inflammation from the injury and harm by non-toxic foreign materials.A part of this study was published in Kinki Daigaku Igaku Zasshi in Japanese as a Ph. D. thesis by Atsuko Ueda. This study was supported by grants from the Ministry of Education, Science and Culture, Japan, the Osaka Eye Bank, Osaka, Japan, and an intramural Research Fund of Kinki University, Japan     

Distribution of blood-borne particles of two different sizes in rat spleens

Polystyrene microspheres 5 micron in diameter and Indian ink, consisting of particles 0.03 micron in diameter, were injected into the splenic artery in rats. The distribution of the polystyrene microspheres and the ink particles in the spleen were examined microscopically and morphometrically. The polystyrene microspheres appeared mainly in the red pulp, and the Indian ink particles mostly in the marginal zone, which functions as an immunological filter. (No arteries opened into the lymphoid follicles.)     

A model of retinal ischemia-reperfusion injury in rats by subconjunctival injection of endothelin-1

The retinal ischemia-reperfusion model is used in the study of transient ischemia-related diseases, such as central retinal artery occlusion, angle-closure glaucoma, and others. There are two methods for experimentally producing an ischemia-reperfusion model in the rat retina: (i) the intraocular pressure is greatly raised by increasing the height of the infusion bottle connected with the needle in the anterior chamber; or (ii) the blood vessel that accompanies the optic nerve in retina is ligated. However, each method has some drawbacks. For example, in the first method, the needle must be fixed in the anterior chamber for 1 hr, thus, the technique is not stable and mechanical damage to ocular structures sometimes occurs. In the second method, because of the unavoidable involvement of the optic nerve, damage to the nerve induces retinal changes unrelated to ischemia. In this study, we injected endothelin (ET)-1 under the conjunctiva of the eyeball (subconjunctival injection), and evaluated whether a retinal ischemia-reperfusion model could be generated by this method, simply and noninvasively. We injected 4 x 10(-5) M ET-1 solution into the right eye of the rat and injected a control vehicle (artificial tears) into the left eye. From 5-60 mins after the injection, 50 mg/ml fluorescein isothiocyanate (FITC)-dextran was injected to the left ventricle of heart. Then, the retina was removed and flat mounted. We compared the perfusion conditions of the FITC-dextran to each retina in the right and left eye. There was a complete perfusion of FITC-dextran in the retinal main artery, vein, and the capillary vessels in all of the control eyes. However, perfusion could not be completely observed in the ET-1 injected eye from 5-35 mins after injection; afterwards, the flow was returned. This method of subconjunctival injection of ET-1 is, thus, a feasible technical option for producing a retinal ischemia-reperfusion model in rat.     

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.