Lissamine Green Research Articles

Effect of histamine on intestinal fluid secretion in the dog.

Intra-arterial infusion of histamine into the small intestine caused about a onefold increase of blood flow, edema of the intestinal tissues and mesentery, and produced a copious secretion of fluid. The jejunal secretions had an ionic composition similar to that of plasma, whereas ileal secretions contained high concentrations of HCO3 with relative low concentrations of Cl. The secretions contained protein (1.5 +/- .2 g/100 ml, range 0.5-2.4) with a similar electrophoretic pattern of plasma protein. When lissamine green was present in the blood, it also appeared in the secretion to a considerable concentration. It is inferred from these findings that a major mechanism of fluid secretion by the action of histamine involves a filtration process across the mucosal epithelium by the incrreased tissue fluid pressure due to extensive capillary leak.

Renal test dyes III. Effect of dyes suitable for renal passage time measurements on renal function.

The effects of the triaryl-methane dyes Lissamine Green SF (LF SF). Lissamine Green BN (LG BN), Lissamine Green V (LG V) and Kiton Pure Blue V (KPB V) on renal functions of rats and on sodium transport across isolated frog skin have been investigated. The experiments failed to show any natriuretic or diuretic effects of the purified dyes on the rat kidney. In the continuous infusion experiments, which lasted 3 1/2 h, the amount of the dye infused was equivalent to a passage time measurement every 3 min. In rapid injection experiments LG V was injected every 5 min for 1 h. The dyes did not have any effect on insulin clearance either. Previously reported effects of the dyes can be partly explained by the presence of sodium sulfate in commercially available dyes. Experiments with LG V showed that the dye also has no effect on the potential difference or short circuit current across the isolated frog skin. This supports their usefulness as renal test dyes.

The role of vesico-ureteric (V-U) reflux in the pathogenesis of kidney scars in the rat.

In Wistar rats V-U reflux with distilled water and sterile saline of varying osmolar strength produces scars in the perihilar region of the kidneys in 80% of animals. Using Indian ink as a marker, rupture of the forniceal epithelium regularly followed V-U reflux. Intrapelvic pressure rose to 120 mm Hg during the reflux procedure. A slowing of proximal tubular transit time was observed by the Lissamine Green technique in the nephrons in the perihilar region immediately after the kidney had been subjected to reflux. It is suggested that the kidney scars which develop after reflux of sterile solutions are ischaemic in origin. It was shown that kidneys damaged by reflux have an increased susceptibility to haematogenous infection.

Quantitative Polyacrylamide Gel Electrophoresis of Crystallin and Solubilized Albuminoid Lens Proteins

Buffer conditions as well as staining and destaining procedures which facilitate a reproducible quantitation of lens proteins in polyacrylamide del electrophoresis (PAGE) were investigated and optimal conditions were chosen. Photopolymerization was found to cause an immobilization of ca 40% of lens proteins in the sample gels. Consequently, PAGE was made with stacking and separating gels only. The dye uptake (Lissamine Green B) to protein ratio was equal for the different proteins in crystallins and solubilized albuminoid. The densitometric recordings of lens proteins within a linear concentration range were resolved into the minimum number of Gaussian distribution functions. These yielded information on the relative content and the mobilities of various lens protein populations. For inbred mouse C57BL lens crystallins a minimum of 26 and for albuminoid a minimum of 20 different proteins were determined. The methods described can be expected to permit a closer insight into lens protein changes with age and disease.

A study of the effects of some chemically welldefined lissamine greens on tubular and overall renal function in the rat (with an evaluation of the validity of lissamine green transit time measurements in studies of tubular function).

AbstractChemically different Lissamine greens were investigated for their immediate and late effects on renal functional parameters. The data indicate (1) that there was no consistent change in transit times (TT) in series of up to four repeated single injections with different LG completed within 8 min. (2) proximal and distal intratubular pressure responded to a single i.v. injection of LG with two distinct pressure increments, similar for all dyes tested. The first increase (prox.: 1–3 mm Hg; dist.: 1–2 mm Hg) coincided with a blood pressure response; the second increase (prox.: 1–2 mm Hg; dist.: 0.5–1.5 mm Hg) started when LG appeared in distal convolutions. The changes in flow velocity resulting from such pressure changes would not cause any significant error in the measured TT. (3) the late effects of LG were an increase in overall reabsorption capacity (predominatly in the loop of Henle) a parallel increase in glomerular filtration rate, and an increase in urine flow and osmotic excretion rate. Proximal reabsorption rate was unaffected. No difference in response was observed between different LG. Neither GFR nor urine flow changed significantly in control experiments in which a similar osmotic activity of mannitol was injected. It is concluded that TT measurements with LG may provide valid information on tubular function; but the late effects of LG would limit its use to a single measurement at the end of an experiment.

Metastases and the normalization of tumour blood vessels by ICRF 159: a new type of drug action.

Profound modification of the structure and arrangement of the blood vessels has been shown in tumours after treatment with ICRF 159. X-ray angiography, carbon black (Pelikan ink) labelling, and intravital staining with lissamine green were used to demonstrate the changes. Alteration of the morphology of the blood vessels at the edge of a tumour may affect the escape of malignant cells and the rate of blood flow (and thus the concentration of anticancer drugs) through the tumour.

The influence of lissamine green on tubular reabsorption of electrolytes and water in rats.

Lissamine Green, administered to rats by infusion into the renal artery or several routes of injection into the jugular vein in various amounts, always produced natriuresis, kaliuresis, and diuresis without change of inulin and PAH clearance. The effect did not depend upon pH or osmolality of the injected solution. The smallest intravenous dose which might still serve for measurement of transit time or identification of tubular segments (0.02 ml of a 5% solution) caused a comparatively small and short change—the colour contrast was weak, however. The mechanism of tubular changes after Lissamine Green is not clear; the shortening of the proximal transit time after repeated injections tends to point to a proximal contribution to the described effect.

MICRO-DETERMINATION OF INULIN CONCENTRATION IN A SOLUTION CONTAINING LISSAMINE GREEN

MICRO-DETERMINATION OF INULIN CONCENTRATION IN A SOLUTION CONTAINING LISSAMINE GREEN

A counter-current system of the surface of the renal cortex of rats.

Using the techniques of Lissamine Green passage and micropuncture on the renal cortex of rats we were able to relate a vascular welling point (branching point of vas efferens) to one nephron. The loops of the nephrons studied on the surface were numbered sequentially in the direction of urinary flow. The distance from each loop to its vascular welling point was measured. As the distance between the loops and their glomerulus increased the distance to their welling point decreased. From these observations it was possible to conclude that the flow of blood was opposite to the urine flow. Under direct observation a counter-current flow was noticed in 72.5% of the peritubular capillaries and tubular loops; in 10.5% the flow was in the same direction, and in 17.0% no predominant direction of flow could be seen (n=131).

Proximal tubular reabsorption in the rat kidney as studied by the occlusion time and lissamine green transit time technique.

AbstractProximal reabsorption rate, determined as the reciprocal of the occlusion time (1/OT), varied in direct proportion to the inulin clearance (Cin) in the range between 1.6 and 0.7 ml/min g KW in nondiuretic rats. Within this range transit time varied from 10 to 24 sec in direct proportion to the occlusion time; the OT/TT ratio averaged 1.10 ± 0.1 (SD). Proximal intra‐tubular pressure averaged 12.4 ± 0.8 mm Hg and did not correlate with CinBelow inulin clearance of 0.7 ml/min g KW transit time (TT) was further increasing from 24 to 44 sec while the OT remained unchanged, thus the OT/TT ratio was decreasing. Proximal intratubular pressure averaged 11.5 ± 0.3 mm Hg.Previous estimates of proximal luminal radius from photomicrographs are revised and a more correct value of about 14 μ was found. The length of the proximal segment, as measured in microdissected nephrons, was 10.4 and 10.5 mm in superficial and juxtamedullary nephrons, respectively. Reflux from more distal segments represented an error of no more than 12 % in estimation of proximal reabsorption rate from the measured OT.The relationship between OT, TT, linear velocity of flow, and tubular fluid/plasma inulin ratio (F/Pin) at 60 % proximal length are derived in quantitative terms. OT/TT = l/ln(F/Pin). From the corrected OT/TT ratio the F/Pin ratio was calculated from values obtained in control rats (“state A”), in rats with partial constriction of the renal artery (“State B”), and in rats with elevated intratubular pressure and urine flow (“State C”).

Concentration of Lissamine green in proximal tubules of antidiuretic and mercury poisoned rats and the permeability of these tubules.

By coloring castor oil with Sudan-red we were able to modify the split droplet method so that concentrations of Lissamine green (LG) could be determined by microphotometry in individual proximal tubular loops of rat kidneys. In the course of the split droplet experiments an increase in the concentration of LG occured, which corresponded to the decrease in volume of the solvent (calculated from the intratubular diameter and the length of the test droplet) only with test lengths of over 100 μ. The change in concentration at shorter lengths of test droplets may have resulted from an incomplete blockage between the oil block and the test droplet.

Individual nephron function in experimental bilateral pyelonephritis. I. Glomerular filtration rate and proximal tubular sodium, potassium, and water reabsorption.

Abstract The function of individual cortical nephrons was studied by micropuncture methods in rats with bilateral retrograde pyelonephritis. It was found that the lumina of tubules in grossly diseased areas were usually occluded and tubular fluid could not be aspirated with a micropipette. Intravenously injected Lissamine green did not appear in the lumina of such tubules. These findings suggest that the severely damaged nephrons were not contributing significantly to urine formation. In adjacent, more nearly healthy areas, GFR per nephron ranged from normal to markedly supernormal values. Proximal tubular reabsorption of sodium, potassium, and water was approximately the same as in normal rats studied under comparable experimental conditions. While it is not possible to conclude with certainty that there were no intrinsic disturbances in proximal transport mechanisms in the surviving nephrons, any such abnormalities were not of sufficient magnitude to be detected by the methods used. The observations are consistent with the view that the major contribution to urine formation in the pyelonephritic kidney is made by nephrons which have undergone adaptational increases in function. Although there are several important differences between the experimental renal disease produced in this study and chronic atrophic pyelonephritis in man, the basic similarity of the histologic features suggests to us that the present observations on nephron function may be relevent to the situation in patients with pyelonephritis.

Observations on periodic acid-Schiff treatment of serum proteins on filter paper with particular reference to counterstaining with Lissamine Green

Observations on periodic acid-Schiff treatment of serum proteins on filter paper with particular reference to counterstaining with Lissamine Green

Elution of Lissamine Green from Cellulose Acetate Electrophoresis Strips

Elution of Lissamine Green from Cellulose Acetate Electrophoresis Strips

Dye binding by protein as applied to quantitative paper electrophoresis

1. (1) The binding of acid dyes by denatured protein on filter paper has been investigated with special reference to Light Green. 2. (2) The uptake of dye, measured by elution, has been shown to vary according to the method of denaturation, the nature of the dye solvent (particularly the salt and and ethanol concentration) and finally with the duration and temperature of the subsequent washing procedure. 3. (3) Albumin and globulin do not behave in the same manner with different denaturing agents so that different methods give different albumin/globulin dye uptake ratios. 4. (4) The stoichiometric relationship between dye and protein is consistently nonlinear even at lower concentrations of protein on the paper and the same pattern can be demonstrated with protein on cellulose acetate membrane. It is suggested that this non-linear relationship at low protein densities is due to interference by the cellulose supporting medium and other anions. 5. (5) Intrinsic constants and the number of binding sites at infinite dye concentration have been measured under standardized conditions for Light Green, Brom-Cresol Green and Lissamine Green. The results have been compared and differences discussed. 6. (6) In the quantitative measurement of protein based on dye uptake, the importance of restricting the protein density on the paper is stressed.

A rapid method for studying tumour blood supply using lissamine green.

Goldacre and Sylven1 have described a method for making visible those regions of solid malignant tumours supplied with flowing blood. They used a high blood concentration of the triphenylmethane dye, lissamine green (Gurr), which turned the whole animal green except where there were barriers. Ten to twelve days following the transplantation of sarcoma 37, mammary carcinoma and Erhlich Landschutz tumours in mice and Walker sarcoma in rats it was observed that vascular obstruction occurred, confining the flowing blood to a thin surface layer of the tumour. Tumours measuring 1–3 cm. across had a white centre and a green periphery. The dye does not penetrate the living cell membrane but the surrounding tissues become green due to the green blood in them and the green interstitial fluid derived from the blood. Extensive regions not supplied with flowing blood but with a thin coating layer supplied with blood were also found in various large spontaneous and methylcholanthrene-induced tumours of rodents. Cells dissected out from the non-coloured tumour region were found to be dead except when within about 300µ from the green region, where mixtures of living and dead cells were found.

Lissamine green as a protein stain in paper electrophoresis

Lissamine green has been tested as a protein stain for use in paper electrophoresis. Nine criteria were enumerated and investigated. 1. (1) The seven batches of lissamine green tested behaved similarly in every respect, as also did one batch tested after repeated use. 2. (2) Lissamine green stains protein but leaves the paper perfectly white. 3. (3) At concentrations in which they occur in paper electrophoresis the dye-uptake of albumin, /gg-globulin, and the mixed proteins of whole serum, is identical. 4. (4) The dye uptake of albumin is proportional to concentration to the maximum investigated (14.23 · 10 −4 mg/mm 2). This is not true of γ-globulin or whole serum, but the relationship remains linear up to the maximum concentration likely to occur in practice. 5. (5) Elution of lissamine green is quick and simple and, although not quite complete, is the same for all proteins within the working range of concentrations. 6. (6) Lissamine green is stable in solution at pH 6.0 and below. There is no need to use a higher pH. 7. (7) Scanning with a simple densitometer does not produce the same results aselution. When the A/G ratio is known, however, the values obtained by densitometry can be corrected to give results corresponding closely to those obtainable by elution. 8. (8) Lissamine green is insoluble in the clearing fluid used for densitometry and in ether used to remove it. 9. (9) The Lissamine green staining technique described is quick, cheap, and simple.