Cresyl Fast Violet Research Articles

Reduced N-Acetylaspartate Levels in Mice Lacking Aralar, a Brain- and Muscle-type Mitochondrial Aspartate-glutamate Carrier

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

A morphometric study on the early stages of dendrite changes in the axotomized motoneuron of the spinal cord in newborn rats

Objectives: To study the early effect of axotomy on spinal motoneurons and dendritic trees in the newborn rat.Methods: The left sciatic nerve of 50 neonatal Sprague–Dawley rats aged 5 days old was transected and the right area kept as a control. The operated animals were killed 2, 4, 8, 12 and 24 hours after axotomy. The L4–L6 segments of the spinal cord were sampled, and stained either with Golgi-Cox or Cresyl fast violet methods. The number of motoneurons, the largest soma diameter and the different parameters of the dendritic trees such as: number, length and thickness of the primary, secondary and tertiary branches in the axotomized sides were estimated and compared statistically with that of the intact sides.Results: The results indicated that in the axotomized sides, the number of motoneurons and the largest soma diameter were decreased, but these were significant only in 12- and 24-hour groups. The number of branches of the dendritic trees including primary, secondary and tertiary branches was not significantly decreased in the groups. The thickness of the dendrites showed a reduction; however, this was significant only for the tertiary branches in the 24-hour groups. The length of the primary, secondary and tertiary branches of the dendrites—especially the latter—were also decreased significantly in most of the groups.Conclusion: Axotomy at the early stages in newborn rats resulted in noticeable morphometrical changes in motoneurons and their dendrites.

Topographic morphology and age-related analysis of the neuronal number of the rat intracardiac nerve plexus

The study was designed to determine the three-dimensional organization of the rat intrinsic cardiac neural plexus (ICNP) and to ascertain whether the rat heart undergoes a decrease in neuronal number with aging as has been reported for other mammalian species, including human. Juvenile (3-4 weeks of age, n = 14) and adult (more than 2 months of age, n = 23) animals were examined using enzyme histochemistry for acetylcholinesterase in order to visualize the ICNP in total hearts. The number of intrinsic cardiac neurons was estimated by counting nerve cells in serial sections of the atrial pieces stained with cresyl fast violet. The total number of intrinsic cardiac neurons in old rats was 6576 +/- 317. The juvenile animals contained significantly fewer such neurons, only 5009 +/- 332. Approximately 70% of all intracardiac neurons were amassed within the heart hilum, while 30% of the neurons were distributed epicardially. Within the interatrial septum, only 11 +/- 11 neurons were identified in the juvenile and 6 +/- 4 neurons in old rats. Extrinsic nerves entered the rat heart in both the arterial and venous parts of the cardiac hilum. The nerves from the arterial part of the cardiac hilum extended directly to the ventricles but the nerves from the venous part of the hilum formed a particular nerve plexus of the cardiac hilum on the heart base. Within the rat epicardium, intrinsic nerves clustered into six routes by which they selectively projected to different atrial and/or ventricular regions. In conclusion, this study provides a detailed description of the three-dimensional organization of the rat ICNP and contradicts the decrease in neuronal number with aging in the rat heart.

Cellular phenology of annual ring formation ofAbies balsameain the Quebec boreal forest (Canada)

Cell formation in growth rings of balsam fir (Abies balsamea (L.) Mill) in the boreal forest was studied to describe the timing of ring formation and the development patterns of earlywood and latewood. Wood micro-cores were extracted during the growing season from 1998 to 2000. The micro-cores were stained with cresyl fast violet to facilitate counting the number of cells in the radial enlargement, wall thickening phases, and mature cell phases. The periods required to complete these various phases were then estimated. Variations in the beginning of the growing season (May 7 – June 7), the earlywood–latewood transition (July 2 – July 19), and the end of the growing season ( August 20 – September 20) were observed. Short cell enlargement durations of less than a week for earlywood and 5–10 days for latewood were observed. Time required for cell wall thickening was about 20 days for earlywood and longer than 10–15 days for latewood. A certain flexibility was observed in the ring formation patterns and in the cell development rate, providing an advantage in the boreal forest where optimal growth conditions change from year to year. These findings on the spatial and temporal patterns of ring development may be useful for understanding tree relationships with climate or other environmental parameters.

Application of the Gompertz equation for the study of xylem cell development

Summary To assess daily relationships between xylem development and the environment, precise knowledge of tree-ring development is required. This includes accurate estimations of the rate and duration of the cell enlargement and wall thickening phases. This paper presents an application where the Gompertz equation is used to calculate cell number increase and to estimate both the rates and periods of the differentiation phases on a daily scale during the growing season. Tests were performed on two coniferous species, Abies balsamea (L.) Mill. and Pinus cembra L., growing in the Canadian boreal forest and on the Italian Alpine treeline respectively. Wood micro-cores, including the most recent tree-rings, were collected during the growing season of 2001. Cross-sections were cut with a microtome and stained with cresyl fast violet to differentiate xylem cells of the growing tree-ring. The number of cells within the zones of radial cell enlargement, secondary wall thickening and mature xylem were counted. The normalised cell number increases were fitted into a Gompertz function [y=f(t)]. The results showed that the function provided suitable descriptions of the cell increase for both Abies balsamea and Pinus cembra with R2 ranging between 0.60 and 0.90. Subsequently, to assess the development phases in time, the Gompertz equation was expressed in function of the independent variable [t=f(y)]. The use of only one equation for the estimation of both cell division and differentiation throughout the vegetative season has demonstrated to be an important improvement.

Study of the nervous tissue development in rainbow trout (Oncorhynchus mykiss) embryos treated with oxytetracycline.

The purpose of this study was to discover whether the use of different doses of oxytetracycline causes any alteration in the development of nervous tissue in rainbow trout embryos. Five thousands eggs of females rainbow trout were divided into five groups. One group acted as control and the other four were administered with one of four doses of oxytetracycline, 0.025, 0.050, 0.100, or 0.201 microM, at the moment of fertilization. The eggs were incubated under pisciculture conditions to just before being ready to spring off. From the 10th day, 10-egg samples were taken regularly and fixed. Five were processed for histological techniques and stained with haematoxylin and eosin, cresyl fast violet and silver, the other five were homogenized for antibiotic detection. Histological alterations appeared in 37-day-old embryos, with an abnormal migration of the neuroblasts to the marginal layer of the neural cord, and alterations in the development of the lens and eye layers. Some embryos showed abnormal curvature of the spinal cord but these changes were not statistically significant.

Sweeping on a microchip: concentration profiles of the focused zone in micellar electrokinetic chromatography.

On-line sample concentration by sweeping was investigated in microchip micellar electrokinetic chromatography (MEKC), By changing the distance between the injection cross and the detection points, the profile of the concentration process and the diffusion process in sweeping was elucidated. Rhodamine B injected for 4 s was best concentrated by sweeping at 9.4 mm from the injection cross and the enhancement factor was 450. At the longer distance from this point the peak of Rhodamine B was broadened and diluted by diffusion. The diffusion constant of Rhodamine B calculated from the experiment was 5.7 x 10(-6) cm2s(-1). The mixture of rhodamine B, sulforhodamine B, and cresyl fast violet was concentrated by sweeping and separated by MEKC at the same time.

Assessment of white matter injury following prolonged focal cerebral ischaemia in the rat.

The ability of putative neuroprotective compounds to protect against white matter injury remains poorly investigated due to the lack of suitable methods for assessing white matter injury. This study was therefore designed to investigate the utility of Tau 1 (oligodendrocytes/axons), myelin basic protein (MBP; myelin) and amyloid precursor protein (APP; axons) immunohistochemistry in assessing white matter injury at various times following middle cerebral artery occlusion (MCAO) in the rat. Focal cerebral, ischaemia was induced in halothane-anaesthetised rats using an intraluminal thread model. At 24 h, 1 and 2 weeks following MCAO, white matter injury was assessed using Tau 1, APP, MBP and Luxol-fast blue staining and neuronal injury with cresyl fast violet (CFV). In histologically normal tissue MBP immunoreactivity was detected in myelinated fibre tracts, while Tau 1 and APP were axonally located. At 24 h following permanent MCAO, MBP, and Tau 1 staining remained relatively unchanged within the myelin and axonal compartments of the ischaemic region. In contrast, increased Tau 1 staining was apparent in oligodendrocytes within ischaemic tissue, while APP accumulated in axons surrounding the lesion. At 1 and 2 weeks following transient MCAO, Tau 1 and APP staining was markedly decreased within ischaemic tissue. Marked reduction in MBP levels within ischaemic tissue were not detected until 2 weeks following MCAO. The area of axonal injury as assessed by reduced Tau 1 or APP staining correlated with the area of neuronal damage as assessed by CFV staining. This study shows that MBP, Tau 1 and APP immunohistochemistry can be utilised to assess myelin and axonal integrity following sustained ischaemia using standard image analysis techniques.

Development of an integrated single-fiber SERS sensor

An integrated fiberoptic surface-enhanced Raman scattering (SERS) sensor based on a single-fiber system is described and evaluated for remote and in situ measurements. The heart of the sensor consists of a unique single optical fiber designed to induce the SERS effect through the incorporation of alumina nanoparticles and silver coatings onto the probe tip. The unique design based on single-fiber for excitation/emission (SFEE) is practical for the fabrication of in situ sensors in microenvironments. Since the single fiber carries both the laser excitation and SERS signal radiation, optical parameters of the remote tip are simple and consistent. Depending on the tip size, the fiberoptic SERS sensor allows in situ measurements at microscale environments. Details of sensor tip fabrication and optical system design are described. Two detection schemes can be used: a “dip-and-dry” mode of detection and an in situ sensing mode are demonstrated for several compounds, including cresyl fast violet (CFV), brilliant cresyl blue (BCB), p-aminobenzoic acid (PABA) and 3-aminopyrene (3AP). To illustrate the effectiveness of this new SERS sensor for remote in situ analysis, SERS spectra of aqueous dye samples (CFV and BCB) are presented. A quantitative calibration curve for CFV in groundwater is established and a limit of optical detection of approximately 50 ppb CFV in groundwater is determined.

A new surface-enhanced Raman scattering substrate based on silver nanoparticles in sol-gel

We report for the first time the development of a new surface-enhanced Raman scattering (SERS)-active silver chloride silica–glass substrate prepared by using in situ precipitation of silver chloride particles in sol–gel films as a precursor for nanoparticles of silver. The controlled precipitation of silver chloride was achieved by the reaction of silver nitrate with trichloroacetic acid, which leads to a slow release of chloride ions. Silver chloride particles were reduced to silver nanoparticles by FeSO4·7H2O. The sol–gel films prepared exhibit good optical properties and induce a strong SERS effect for several model compounds, including cresyl fast violet and brilliant cresyl blue. Optimization studies of substrate preparation and coating conditions were performed and are discussed in detail. The performance of this new substrate compares favorably with those of previously developed silver-coated alumina substrates. The SERS spectra of other compounds of environmental and biological interest, such as 1−aminonaphthalene, 2−aminopyrimidine, pyridine and p$-aminobenzoic acid, were investigated to illustrate the usefulness of this novel type of SERS substrate for use in chemical and biological analysis. Copyright © 1999 John Wiley & Sons, Ltd.

Spectrophotometric Estimation of Nimesulide and its Formulations

Four simple and sensitive visible spectrophotometric methods (A–D) have been described for the assay of nimesulide (NMD) either in pure form or in pharmaceutical formulations. Methods A and B are based on the oxidative coupling between the reduced product of NMD (RNMD) and p-N,N-dimethyl phenylenediamine dihydrochloride (DMPD) in presence of chloramine – T (CAT) or 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in presence of ferric chloride (Fe III) to form coloured products with λmax at 540 nm and 600 nm respectively. Method C is based on the diazotization of RNMD with excess nitrous acid (HNO2) and estimating the consumed HNO2 with cresyl fast violet acetate (CFVA). Method D is based on the formation of the coloured charge-transfer complex, when RNMD is treated with metol (p-methyl aminophenol sulphate, PMAP) in presence of potassium dichromate. All variables have been optimized and the reaction sequence is presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%. Recoveries are 98.6–100.2%.

Spectrophotometric determination of azathioprine in pharmaceutical formulations

Four simple and sensitive visible spectrophotometric methods (A–D) have been described for the assay of azathioprine (ATP) either in pure form or in pharmaceutical formulations. Methods A and B are based on the oxidation of ATP with excess N-bromosuccinimide (NBS) or chloramine-T (CAT) and determining the consumed NBS or CAT with a decrease in colour intensity of celestine blue (CB) (method A) or gallocyanine (GC) (method B), respectively. Methods C and D are based on the diazotisation of reduced azathioprine (RATP) with excess nitrous acid and estimating either the consumed nitrous acid (HNO 2) with cresyl fast violet acetate (CFVA) (method C) or by coupling reaction of the diazonium salt formed with N-1-naphthyl ethylene diamine dihydrochloride (NED) (method D). All of the variables have been optimized and the reactions presented. The concentration measurements are reproducible within a relative standard deviation of 1.0%. Recoveries are 99.2–100.3%.

Near-field surface-enhanced Raman spectroscopy of dye molecules adsorbed on silver island films

We report the combined use of scanning near-field optical microscopy and surface-enhanced Raman scattering (SERS) to obtain spectral, spatial and chemical information of molecular adsorbates with subwavelength lateral resolution. Near-field SERS spectra of cresyl fast violet and rhodamine 6G on silver substrates are obtained with short exposure times (less than 100 s) and a signal-to-noise ratio of >20. Spectra from as few as ≈300 molecules or less than 10 −2 monolayers adsorbed on about ten silver nanoparticles can be recorded using 200 nm tip apertures. For the near-field spectra, a local Raman enhancement factor of 10 13 or greater can be derived from a comparison with fluorescence measurements.

Application of Azine Dyes for the Determination of Ranitidine Hydrochloride in Pharmaceutical Formulations

Utilisation of azine dyes for the development of four simple spectrophotometric methods are described for the determination of Ranitidine hydrochloride (RTH) in pure form and pharmaceutical formulations. Method A is a direct one, which involves the formation of an ion-association complex (λmax:600 nm) between the drug molecule and wool fast blue BL. The other three methods, indirect ones, involve the addition of excess oxidant (chloramine-T, for method B; potassium permanganate, for method C, N-bromosuccinimide, for method D) of known concentration in acid medium to RTH and the determination of the unreacted oxidant by measurement of the decrease in absorbance of gallocyanine (GC for method B), cresyl fast violet acetate (CFVA for method C) or celestine blue (CB for method D) at a suitable maximum wavelength (λmax:540 nm for methods B&D and λmax: 555 nm for method C). The validities of the methods are tested against the spectrophotometric reference method and all are in good agreement.

Spectrophotometric Determination of Bio-Active Compounds in Commercial Samples with Nitrous Acid and Cresyl Fast Violet Acetate

Abstract A simple and sensitive method for the spectrophotometric determination of bio-active compounds (drugs and non-nutritive sweeteners) containing reactive functional groups, viz., aromatic primary amine (drugs: Dapsone, Sulphamethoxazole), aromatic secondary amine (drug: Pindolol), aliphatic secondary amine (non-nutritive sweetener: Cyclamate), acid hydrazide (drug: Isoniazid) and thiol (drug: Captopril), is proposed. The method involves the addition of excess of sodium nitrite to the compound in the presence of 0.25 M hydrochloric acid solution and the unreacted nitrous acid is determined by the measurement of corresponding decrease in the absorbance of cresyl fast violet acetate (Δmax : 555 nm), the most suitable one out of several dyes tested. This method was applied for the determination of bio-active compounds in commercial samples. (drugs: pharmaceutical formulations; non-nutritive sweetener (cyclamate): foodstuffs). The newly proposed method enabled the determination of the bio-active compoun...

Screening and specification of colour changes of nitritometric indicators

The specification of colour changes of nitritometric indicators, viz., cresyl fast violet acetate, amethyst violet, safranine O, neutral red, methylene violet, neutral violet, phenosafranine, brilliant cresyl blue and lissamine blue BF, in the titration of sulphanilamide has been carried out with the help of tristimulus colorimetry. Apart from the determination of true colour co-ordinates and complementary colour co-ordinates, the quantitative parameters specific colour discrimination (SCD), total colour difference (ΔE*) values of CIE La*b*, CIE Lu*v* and LAB HNU colour spaces together with the standard deviation of colour matching (ΔS) were also evaluated for all the indicators at different concentrations ratios of nitrite and sulphanilamide, for a quantitative measure of quality of colour change. The quality of colour change of many of the studied indicators is further improved by screening with appropriate inert colorants to obtain a colour transition between complementary colours through colourlessness at the equivalence point.

Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope.

A simple modification of the physical disector is presented which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 microm thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections in investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.

Stereological estimation of the total number of neurons in the asexually dividing tetrathyridium of Mesocestoides corti

SUMMARYFifty tetrathyridia of Mesocestoides corti showing various degrees of scolex fission during asexual multiplication were subjected to estimation of the total number of neurons in the central ganglia, using a ‘fractionator’ designed sampling scheme on every 10th Historesin section through the specimen. Neurons were stained with Cresyl fast violet and viewed by transmission light microscopy. Cells were only counted if a nucleolus was present. The animals were divided into groups according to their stage in the asexual reproduction process indicated by head shape and sucker number. Animals which had a narrow head with 4 suckers contained about 250 neurons. As their heads became wider, in preparation for fission, neurons were overproduced by up to 5-fold. As additional suckers were developed (8 in total), neurons were reduced to about 500. Finally, when the animals had 2 clearly discernible heads, with 4 suckers on each, the total number of neurons (per head) was again approximately 250. As the heads became wider again, neuron over-production recommenced. Exceptionally shaped animals with an X- or T-shaped single head were observed. These results suggest that there is a preparatory stage for neuronal regeneration, during which the basic population of neurons is about 250. This is followed by neuron over-production followed by a selective cell death of neurons which occurred during asexual multiplication. Another cycle for neuronal regeneration can occur before an animal completes its fission process.

Staining myelin and myelin-like degradation products in the spinal cords of Chronic Experimental Allergic Encephalomyelitis (Cr-EAE) rats using Sudan Black B staining of glycol methacrylate-embedded material

A high-resolution light-microscopical (HRLM) technique is described to visualize myelin, and macrophages containing degradation products of myelin, in the spinal cords of chronic relapsing experimental allergic encephalomyelitis (Cr-EAE) rats. This HRLM technique was developed to optimalize the correlation between nuclear magnetic resonance (NMR) characteristics and histopathological images in this well-established animal model for multiple sclerosis (MS). Spinal cords were fixed by perfusion with a combination of cacodylate-buffered glutaraldehyde and formaldehyde, post-fixed in Dalton's fixative (containing osmium tetroxide), rinsed in water, processed in ethanol, acetone, and embedded in glycol methacrylate resin (Technovit 7100/HistoResin). Semi-thin sections were stained with Sudan Black B and counterstained with Cresyl Fast Violet, resulting in black staining of myelin and its degradation products, with blue/violet staining of demyelinated axons and other tissue elements. These dyes were selected with the aid of a numerical model of staining, which took both access and lipophilicity into account. The staining procedure is simple and highly reproducible. The resulting images are contrast rich, and combine excellent morphology with a high degree of lipid retention.

The effect of age and Alzheimer's disease on pyramidal neuron density in the individual fields of the hippocampal formation.

Pyramidal neuron density was determined at autopsy in the brains of 12 neurologically normal patients (age range 6-87 years) and 18 patients with histopathologically confirmed Alzheimer's disease (AD; age range 62-89 years). Paraffin wax sections were cut at the level of the central part of the cornu ammonis and stained with cresyl fast violet. Pyramidal neuron density was determined in the stratum pyramidale of the presubiculum, subiculum, prosubiculum and CA1-4. The width of the stratum pyramidale in these areas was also determined. There was no significant effect of age on pyramidal neuron density in any of the fields of the hippocampal formation. However, there was a significant decrease in pyramidal neuron density in the subiculum (44%), prosubiculum (28%) and CA1 (41%) of AD brains compared to controls. There was no significant effect of AD on pyramidal neuron density in the presubiculum, CA2 and CA3, but in CA4 it increased (23%) significantly. Pyramidal band width decreased significantly with age in the presubiculum but there was no effect of age on any other region of the hippocampal formation investigated. The width of the stratum pyramidale was significantly lower in the presubiculum (19%), subiculum (30%) and CA3 (17%) of AD compared with control brains. These data suggest that a reduction in pyramidal neuron density within the hippocampal formation does not occur in the absence of underlying pathology. In AD, pyramidal neuron loss predominantly occurs from that sector of the hippocampal formation which comprises the subiculum, prosubiculum and CA1.