Effect of phosphates on the disintegration of red blood cells by brilliant cresyl blue in correlation to age

The author describes age-related changes in the disintegration of rat red blood cells (RBC) caused by brilliant cresyl blue (BCB) in isotonic saline containing NaCl and phosphates. Blood was taken from rats aged 21, 45, 90-105, 340-360 and 690-720 days. The RBC were incubated four hours in the given solution. The incubation of RBC from different age groups in isotonic saline with phosphates showed no significant differences between the various age groups as far as disintegration of the RBC was concerned. When BCB was added to the solution, however, there was a marked increase in the disintegration of RBC from 21-, 42- and 690- to 720-day-old rats. The differences found in RBC disintegration can be attributed to age-determined qualitative changes in the structure of the RBC membrane--in the first place, changes in cross-linkages between the chains of the structural proteins of the RBC membrane.     

Influence of acute hypoxia upon brilliant cresyl blue-induced disintegration of erythrocytes in rabbits and cats

The disintegration of erythrocytes by brilliant cresyl blue was studied in rabbits and cats during two hypoxic periods resulting in apnoea which was followed by artificial ventilation and recovery. The rate of erythrocyte disintegration was measured after 2 hours' incubation in isotonic NaCl or Krebs-Ringer solution plus brilliant cresyl blue (0.5 mmol.l-1). Significant increases of disintegration rates were found in rabbits during recovery in both incubation solutions. Erythrocytes in cats seemed to be more resistant to acute hypoxia, as their disintegration rate rose only in isotonic NaCl solution, and that only transiently. Brilliant cresyl blue--induced erythrocyte disintegration in cats did not differ from the control values during 10 min of spontaneous breathing after the first and the second period of hypoxia in the isotonic NaCl solution, but it was significantly lower in Krebs-Ringer solution. The possible factors influencing the erythrocyte disintegration rate in acute hypoxia are discussed.     

Multinucleated erythroid cells in blood of golden hamster embryos

Blood smears of 11 and 13-day-old embryos (20) and newborns (10) of golden hamsters were analysed. The smears were stained with brilliant cresyl blue in a moist chamber for 5--7 min. About 2--6 thousand cells were counted in 200 visual fields. By means of an ocular micrometer MOB-1 the diameters of 300 cells were measured and the curves on cell distribution were plotted according to the diameters. It was found that the blood of 11-day-old golden hamster embryos contained about 8% of binuclear and sixnuclear megaloblasts, and that of 13-day-old embryos--about 2.5% of binuclear and sixnuclear erythroblasts and megablasts of the second generation. In the bloood of newborn hamster polynuclear cells are absent.     

Correlation of isonicotinic acid hydrazide infiltration across the blood-brain barrier to postnatal development in rats.

Changes in the isonicotinic acid hydrazide (INH) concentration in rat blood and brain were studied in correlation to postnatal development in groups of animals aged 21 and 42 days and 3 months. In the first part of the experiments, INH was administered intravenously to all the age groups in a dose of 100 mg/kg. In the second part, the dose was related to extracellular fluid volume, so that the 3-week-old rats were given 154 mg/kg, the 6-week-old animals 129 mg/kg and the 3-month-old animals 100 mg/kg. After a dose of 100 mg/kg, INH levels in the blood of 21-day-old rats were significantly lower than in 42-day-old and adult animals. The brain INH levels did not differ significantly. On relating the dose to the amount of extracellular fluid, there were no significant differences in the blood INH levels, but the levels in the brain of 21- and 42-day-old rats were significantly higher than in 3-month-old animals. Blood volume related to body weight and brain weight did not differ in the various age groups. The authors conclude that the blood-brain barrier for isonicotinic acid hydrazide alters in rats during postnatal development. In young animals (21- and 42-day-old), more INH infiltrates into the CNS than in adult animals.     

Age-correlation of protein utilization to saccharide intake

Male Wistar rats aged 30, 90, 150 and 360 days were fed ad libitum on diets with an optimum protein and fat content for their respective ages and an increasing saccharide content. Net protein utilization (NPU) was determined from the body nitrogen and protein intake values and the course of gluconeogenesis in the liver was measured by specific phosphoenolpyruvate carboxykinase (PEPCK) activity. According to the growth curve for the standard diet, animals aged 30 and 90 days have a high growth rate (3.245 g/day), 150-day-old rats grow more slowly (1.856 g/day) and 360-day-old animals put on scarcely any weight at all (277 mg/day). In 30-day-old rats, NPU attains maximum values in the presence of a 36% saccharide content in the diet, in 3- and 5-month-old animals in the presence of 51% saccharides and in one-year-old animals in the presence of 41% saccharides in their food. The course of gluconeogenesis also corresponds to these values. PEPCK activity in the youngest age group is greatest in the presence of 31% saccharides in the food, at 90 days it is stimulated in the presence of 31-46% saccharides, at 150 days the decisive concentration is 41 and 46% and at one year proteins are used for saccharide synthesis in diet with a 31 and 36% saccharide concentration. For optimum saccharide values, PEPCK activity is reduced in every age group; together with the maximum NPU values, this indicates that proteins are used for growth and building of the organism at an early age and for the renewal of tissues and organs and maintenance of the organism in adulthood.     

Flow cytometric analysis of blood cells stained with the cyanine dye DiOC1[3]: reticulocyte quantification

The fluorescent dye 3,3'-dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes less than immature erythrocytes congruent to platelets less than leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, new methylene blue, brilliant cresyl blue (BCB), and acridine orange (AO). However, the population of imRBC as quantified by DiOC1[3] fluorescence is a subset of reticulocytes (reticulocytes as determined by BCB assay). The advantages and disadvantages of using DiOC1[3], AO, or pyronine Y as reticulocyte stains are discussed.     

Automated cytometry of fibre size and spatial distribution in the superficial masseter muscle of the rat at three ages

Summary An image cytometry program was applied to sections of the superficial masseter muscle of female and male 21-, 42- and 105-day-old rats. Lesser diameter and spatial distribution of more than 6000 muscle fibres were automatically measured in digital images from muscle sections stained for myofibrillar ATPase activity.In this muscle, only type 2A, 2B and 2C fibres were observed. At the three ages and in both sexes, 2A fibres were found to have the largest diameter and were the most frequent (> 54%). In the 21-day-old animals, females presented larger diameters than did males; in contrast, in the 105-day-old animals, the three fibre types were larger in males than in females. At all ages and in both sexes, type 2A occupied 32 to 80% more cross-sectional area than type 2B. Most images (98%) showed a random spatial distribution of their fibre types.All fibre types grew in diameter with age. The coefficient of variation of the diameter was age-independent and remained under 23%. The finding of an age-independent variable could have a practical application: an increase of the coefficient of variation (> 25%) can be considered as pathological, even without a perfect age-matched control.     

Age-Related Changes of the Anxiety Level of Male and Female Rats in Elevated Cross-Maze Test

Age-related peculiarities of formation behavior in the elevated cross-maze was studied in male and female Wistar rats of 6 age groups: the 17-, 21-, 30-, and 36-day-old rat pups as well as adult animals. Recorded were duration of animal stay in the open arms of the maze and the number of hanging-down reactions, parameters of the level of anxiety. In the 17-day-old rat pups the anxiety level was higher than the older animals, the 17-day-old pups being characterized by frequent manifestation of freezing reaction. Duration of stay in the open arms of the maze increased in the 26-day-old rat pups. In the 21-day-old animals the number of hanging reactions rose as compared with the 17-day-old ones, but did not differ from that in the 26-day-old animals. The anxiety level in the 30-day-old rat pups was much lower than in animals of younger age groups. In the rats aged 36 days (the age directly preceding sexual maturation) there was a pronounced enhancement of anxiety, recorded using both parameters. In the 42-day-old animals an increased duration of stay in the open maze arms was again observed, which indicates a decrease of the anxiety level. The obtained data demonstrate complex non-linear development of this form of emotional behavior in ontogenesis and allow suggesting an important role of the hormonal-humoral system in its formation.     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.     

Antiphlogistic action of phenylbutazone in normal and adrenalectomized rats of different ages relation to the 5-hydroxytryptamine blood concentration.

The authors have attempted to demonstrate the effect of bilateral adrenalectomy on carrageenin-induced oedema and on the antiphlogistic action of phenylbutazone in relation to the changes of blood 5-hydroxtryptamine (5-HT) in rats of different ages (21 days, 42 days, 3 months and 18 months old). It was found that the influence of adrenalectomy on the antiphlogistic action of phenylbutazone and on the blood 5-HT concentration is related to the age of rats. The lowest antiphlogistic action of phenylbutazone was found in 21-day-old rats and highest in the 18-month-old ones. In adrenalectomized 21- and 42-day-old rats the antiphlogistic action is decreased and fully suppressed in rats 3 and 18 months old. Adrenalectomy does not influenced basal values of blood 5-HT concentration. Blood 5-HT in adrenalectomized rats with inflammationadrenalectomized rats 42 days and 3 months old with inflammation after injection of phenylbutazone an increase of 5-HT was observed, but in 18-month-old animals in which antiphlogistic action is highest a decrease of 5-HT was observed.     

CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

Ophthalmoscopic observations of ocular fundus in colony-born cynomolgus monkeys aged from 0 day to 90 days

Apparently healthy 242 colony-born cynomolgus monkeys (Macaca fascicularis) aged from 0 day to 90 days were examined for the findings of ocular fundus by using ophthalmoscope. One drop of mixed solution of 0.5% tropicamide and 0.5% phenylephrine hydrochloride was instilled into each eye of the monkey. Then, those monkeys were anesthetized with ketamine-HC1 at the dose level of 10mg/kg B. W. Regular and fluorescein photographs were taken with Kowa RC-II ophthalmoscope-camera by using daylight typed color film. Following findings were obtained in each age class: Retinal color was salmon pink with 0 to 3-days-old neonates, salmon pink and blue to green with 7 days to 14-days-old animals and blue to green with 60-days to 90-days-old monkeys. As regards optic disc, 0- to 14-days-old animals were observed to be light orange in color, and the infant aged more than 28-days showed orange color. Retinal arteries and veins were lightly reddish in color with every age class. Macular color was salmon pink in 0-day-old cases, slightly dark in 3-days-old neonates and very dark after 14-days of age. Lightly retinal reflex was noted in 0- and 7-days-old animals. The reflex was observable in 14-days-old animals without any case of exception. Retinal hemorrhages were recorded in 22 (67%) of 36 neonates born in natural condition and 10 (33%) of 30 neonates born by cesarean section. These findings will be useful as the criteria for ophthalmoscopic observations of the cynomolgus monkeys as laboratory use.     

Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogenetic development of convulsant action of Ro 5-3663 in the rat

Motor seizures were induced by Ro 5-3663 in 156 male albino rats aged 7,12,18,25, and 90 days. Both minimal and maximal seizures could be elicited in 18-day-old and older animals, whereas only maximal seizures were induced in the two youngest groups. ECoG changes were studied in other 21 young rats. First changes induced by Ro 5-3663 were formed by isolated sharp waves in 7- and 12-day-old rats and by episodes of rhythmic activity in older animals. An imperfect form of this rhythmic activity could be seen even in 12-day-old rats. Ictal ECoG activity exhibited an increase in frequency of individual graphoelements, in generalization and in synchronization of activity among different cortical regions with maturation. Correlation between motor and ECoG phenomena was poor in 7-day-old rats and ameliorated with age but it never reached perfection. The actions of Ro 5-3663 are identical with those induced by metrazol but they differ from those elicited by bicuculline or 3-mercaptopropionic acid.     

Susceptibility dynamics in neonatal BALB/c mice infected with Cryptosporidium parvum.

BALB/c Mice were infected as neonates and at different ages to study the susceptibility dynamics in this animal model to Cryptosporidium parvum. When 4-day-old animals were infected with 10(5) C. parvum oocysts, parasites were detected in the terminal ileum when the mice became 14-25 days old (10-21 days post-infection [PI]). The percentage of animals positive for parasites was 100% up to the age of 19 days (15 days PI) but decreased immediately thereafter until no parasites were detected in 26-day-old (22 days PI) or older mice. Parasite load also decreased in these animals from 184.7 parasites per high power field in 14-day-old animals (10 days PI) to 0.22 in 25-day-old (21 days PI) mice. In a second study, some neonatal mice became resistant to C. parvum when infection was attempted at day-10 of age (day-15 of age at sacrifice). The susceptibility to C. parvum decreased until 14 days of age (19 days of age at sacrifice) when mice could no longer be infected. Parasite load also decreased in infected mice from 235.6 parasites per high power field (9 days of age at sacrifice) to 0.25 (18 days of age at sacrifice).     

Why inclusion bodies do assume different locations in thalassaemic erythrocytes

The reported scanning (SEM), transmission (TS), and freeze-etching (FE) electron microscopic studies have agreed in confirming that in thalassaemic erythrocytes, previously incubated with brilliant cresyl blue (BCB), the unpaired alpha chains precipitate in the central portions of the cell whereas excess beta chains locate in the submembranous regions. This is due to the fact that beta chains, possessing two thiols instead of only one (as in alpha chains), are more liable to bind to similar groups contained in the inner red cell leaflet. Less soluble alpha chains tend to form inter-chain bridges and thus precipitate centrally. SEM observations have given evidence that on the surface of the affected red cells denaturated alpha chains give rise to large and shallow invaginations whereas denatured beta chains lead to diffuse wrinkled appearance. The causes of the different SEM aspects have been suggested.     

ACCUMULATION OF BRILLIANT CRESYL BLUE IN THE SAP OF LIVING CELLS OF NITELLA IN THE PRESENCE OF NH3

When the living cells of Nitella are placed in a solution of brilliant cresyl blue containing NH₄Cl, the rate of accumulation of the dye in the sap is found to be lower than when the cells are placed in a solution of dye containing no NH₄Cl and this may occur without any increase in the pH value of the cell sap. This decrease is found to be primarily due to the presence of NH₃ in the sap and seems not to exist where NH₃ is present only in the external solution at the concentration used.     

Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests

The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.