Endpoints for Stroke Studies
Yanlin Wang-Fischer in Manual of Stroke Models in Rats, 2008
Evans blue is an anionic dye and is a large molecule closely related to trypan blue. It has been used to measure blood volume because it binds to serum proteins and stays in the circulation for a few hours.78 When it leaves the blood, some of it binds to collagen (its elongated structure favors this), and some is taken into cells, including macrophages and neurons. The dye–protein complex is fluorescent (red emission) and was the first fluorescent tracer of neuronal uptake and retrograde axonal transport. Evans blue can be eliminated from tissue by washing in slightly alkaline water. Evans blue and trypan blue both can be used to determine cell vitality; live cells exclude the dye, dead cells take it up, forming the basis of the trypan blue exclusion test. Evans blue dye has been used for quantitative evaluation of BBB permeability in stroke and in myocardial ischemia.79,80 Infarcted tissue stains blue, but normal tissue does not stain. This technique avoids the costs associated with radioisotope use and is simpler than the [3H] sucrose method since it does not require an integral sample. The only equipment required is a spectrophotometer. However, the Evans blue technique can only produce total permanent values; rate constants cannot be estimated this way.
Animal, Human, and in Vitro Test Methods for Predicting Skin Irritation
Francis N. Marzulli, Howard I. Maibach in Dermatotoxicology Methods: The Laboratory Worker’s Vade Mecum, 2019
In order to distinguish between mild and moderate irritants in an acute exposure test, Finkelstein and his colleagues (1963, 1965) used pretreatment of test sites with an irritant and enhanced visualization of the response by injection of trypan blue in order to increase test sensitivity. The technique was performed in anesthetized rabbits, rats, or guinea pigs. A circular area of the shaved abdomen was painted with a 20% solution of formaldehyde that was allowed to dry for 5 min. This was repeated three times and then 1 -in cotton flannel pads were saturated with test material and applied to each site. A control substance of known irritancy was tested in each study. Pads were secured in place and the entire trunk was wrapped in polyethylene. A solution of trypan blue was injected into subcutaneous tissue away from the dosage sites. The dye was absorbed and served as a marker for plasma leakage because it spontaneously binds to albumin. After 16 h, patches were removed and the degree of bluing at each site was evaluated on a 0–100% scale. In light of more recent work comparing the reactivity of dorsal and abdominal animal skin (Vinegar, 1979), one wonders if the enhanced sensitivity was due in part to choice of test site.
ExperimentaL Oral Medicine
Samuel Dreizen, Barnet M. Levy in Handbook of Experimental Stomatology, 2020
Microscopic reflections of early radiation injury in the rat parotid gland were elucidated by Sholley et al.100 Female Sprague-Dawley rats weighing between 170 and 230 g received either 1600 or 6400 R in a single exposure delivered at a rate of approximately 190 r/min. Trypan blue and colloidal carbon were used as tracers to detect abnormal vascular permeability. Trypan blue was administered in a dose of 0.2 mℓ of a 2% solution in saline per 100 g body weight. The tracers were injected into the lateral tail vein of the anesthetized rats. Some rats received both tracers; most received only one. Circulation time ranged between 10 and 90 min before sacrifice. Parotid glands were examined by light and electron microscopy after appropriate fixing and processing. For light microscopy, the glands were fixed in 10% formalin and prepared for paraffin sections stained with hematoxylin and eosin and with special stains. For electron microscopy, the glands were fixed in Karnowsky’s fixative, postfixed in osmium tetraoxide, dehydrated in ethanol, and embedded in Epon®.
Riboflavin immobilized Fe3O4 magnetic nanoparticles carried with n-butylidenephthalide as targeting-based anticancer agents
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Trypan blue was used to stain the resultant cells. Dead cells allowed trypan blue to penetrate the cell membrane resulting in cells with blue colored stains. Thus, cell viability was visualized under an optical microscope. Trypan blue (0.4%, 0.5 mL) was prepared in an aqueous NaCl (0.81%) and K2HPO4 (0.06%) solution and was mixed with a specific media (0.5 mL). The mixture was added to the cells prepared above and was cultured for 72 h. The samples were incubated in an incubator containing 5% CO2 at 37 °C for 30 min. The resultant cells stained with Trypan blue were rinsed with an aqueous NaCl (0.9%) (1 mL × 3) solution and were fixed by paraformaldehyde (4%) prepared in aqueous NaCl (0.9%) solution for 30 min. The cells were rinsed with aqueous NaCl (0.9%, 1 mL × 3) solution and were treated with a blocking buffer (aqueous solution containing NaCl (0.9%), FBS (10%) and Triton X-100 (0.25%) at 37 °C for 30 min. The nuclei of the cells were stained with Hoechst 33342 dye (2 mg μL−1, 100 μL) and incubated at 37 °C for 30 min. The cells were rinsed with washing buffer containing NaCl (0.9%) and triton X-100 (0.25%) (1 mL × 3). Finally, the cells on glass cover slips were covered with a microscope slide deposited with a drop of glycerol (80%, 20 μL) was sealed with nail varnish. The samples were investigated under an optical microscope.
Assessment of the genotoxic effects of antihypertensive drug active ingredient indapamide in human lymphocytes
Published in Drug and Chemical Toxicology, 2023
Ece Avuloglu-Yilmaz, Deniz Yuzbasioglu, Fatma Unal
Comet assay was performed according to the procedure of Singh et al. (1988). Biocoll was applied to isolate lymphocytes from whole blood. Cell viability was determined using trypan blue. The viability of cells was determined as ≥96%. Lymphocytes were incubated at 37 °C for one hour with four different concentrations of indapamide. A positive (100 μM H2O2), a solvent (5.4 μl/ml methanol) and a negative control was also present. All procedures were made according to Avuloglu-Yilmaz et al. (2020). In all the applications, a total of 300 cells were analyzed under fluorescence microscopy (Olympus BX51; with an excitation filter of 546 nm and a barrier filter of 590 nm) at 400 × magnification using ‘Comet Assay IV,’ Perceptive Instruments Ltd., UK analysis system. DNA damage was evaluated using tail intensity, tail length, and tail moment.
Continuous Curvilinear Capsulorhexis – A Practical Review
Published in Seminars in Ophthalmology, 2022
The lens capsule is colorless, but in the presence of a normal red reflex, the anterior capsule can easily be seen once a flap is created and deflected.4 Adequate visualization of the capsule is a prerequisite for the creation of a proper capsulorhexis.29 Staining the capsule helps visualization but might also stiffen the capsule and make it more vulnerable to tears. Additionally, trypan blue may damage the corneal endothelial cells.5 For these reasons, the use of dyes should be reserved for cases in which visualization is difficult, such as white cataracts, brunescent cataracts, vitreous hemorrhage, and corneal clouding.4,5,29 Capsule staining can also be useful for teaching purposes.5,29 A few approaches are available.4,5 In the most common approach, the stain is injected under an air bubble and washed out after 10 to 15 seconds. Trypan blue is the most often used, and it is the only dye that is approved by the US Food and Drug Administration (FDA).4,5,16,29 In addition, in 2006, the American Academy of Ophthalmology (AAO) reported that there is level III evidence that trypan blue, indocyanine green and fluorescein effectively stain the capsule.29 However, fluorescein is less convenient to use, and indocyanine green has less intensity and is not recommended for brunescent cataracts due to reduced contrast with the lens.5 Gentian violet has been found to provide comparable results to trypan blue in terms of capsule visualization, but it has an inferior safety profile.5
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