Ultrastructure of the periaqueductal grey matter of the rat: an electron microscopical and horseradish peroxidase study.

The neurons of the mesencephalic periaqueductal grey substance (PAG) in the rat are small and medium sized. The cells are frequently located in small clusters, without interdigitating glial elements and may be connected by direct membrane appositions or by gap junctions. The inner zone of the PAG is cell poor. In many cases, the cytoplasm of the cells is filled with extensive rough endoplasmic reticulum, free ribosomes, Golgi apparatus, and large lysosome-like granules. The nuclei show large indentations. The cells have a high nucleus-cytoplasm ratio. The neuropil is very extensive and particularly rich in large numbers of small unmyelinated axons, dendrites, axonal varicosities, and synaptic connections. Myelinated fibres are relatively scarce. The orientation of the fibres was studied in transverse and horizontal sections, in combination with HRP track tracing experiments. It appeared that throughout the PAG most of the fibres were orientated longitudinally. Quantitation showed that most fibres were present in the inner zones of the PAG. Moreover, the diameter of the fibres adjacent to the aqueduct was smaller than that of the fibres in the peripheral parts of the PAG. The thin unmyelinated fibres made extensive synaptic connections within the PAG. Many synaptic varicosities were found in the neuropil of the PAG. There were four types of synaptic varicosities, characterized by different populations of clear and dense-core secretory granules and by the different morphology of the synaptic specializations. In general, the different types of varicosity were homogeneously distributed in the different parts of the PAG. Electron dense secretory granules, when present, were located at some distance from the synaptic junction. Serial sections revealed varicosities which contained only dense-core secretory granules, without synaptic specializations. The dendrites of PAG neurons generally lacked synaptic spines. Many dendrites, particularly those of neurons located in the peripheral parts of the PAG, were directed toward the aqueduct. The present study shows that the PAG is a very complex brain area. The crisscrossing of axons and dendrites with synaptic connections at considerable distances from the cell bodies render it very difficult to unravel the relationships between the possible sources and destinations of ongoing information. This structure complicates the search for relationships between the functional organization and the cytoarchitectural borders in the PAG area.     

A method to quantitate axonal injury

The quantitation of diffuse axonal injury provides a more objective approach to the assessment of tissue damage in head injuries. The method designed in this study takes into account the anisotropy and structural inhomogeneity of the brain, and the distribution of lesions in diffuse axonal injury. The number of counts required for the statistical analysis is inversely proportional to the square of the desired accuracy, specified as the percentage of the mean value of the axonal balloons since the true mean is unknown from the outset. The number of fields are examined using an indexed-squares graticule in 10 different areas of the brain. Silver-stained sections from the brains of head injured patients that survived longer than 12 h must be used with this method. Difficulties may arise when patients of different survival times are compared since it takes some time for the axonal balloons to develop. A correlation with the survival time can be established with the quantitative data collected. The morphometric principles and the statistical rationale on which this methodology is based are briefly presented.     

Quantitative proton MR spectroscopic imaging of the human brain

Multislice proton MR spectroscopic images (SI) of the brain were quantitated, using the phantom replacement technique. In 16 normal volunteers, ranging in age from 5 to 74 years, average “whole brain” concentrations of choline (Cho), creatine (Cr), and N-acetylaspartate (NAA) were found to be 2.4 ± 0.4, 7.9 ± 1.3, and 11.8 ± 1.0 (mM, mean ± SD), respectively. These values are in good general agreement with those previously determined by single-voxel localization techniques. Cortical gray matter was found to have lower Cho and NAA levels, compared to those of white matter, corpus callosum, and basal ganglia. Cho was also found to increase significantly with age in several locations. Quantitative multislice proton SI is feasible in the clinical environment, and regional and age-dependent variations occur that must be accounted for when evaluating spectra from pathological conditions.     

An algorithm for the automated quantitation of metabolites in in vitro NMR signals.

The quantitation of metabolite concentrations from in vitro NMR spectra is hampered by the sensitivity of peak positions to experimental conditions. The quantitation methods currently available are generally labor intensive and cannot readily be automated. Here, an algorithm is presented for the automatic time domain analysis of high-resolution NMR spectra. The TARQUIN algorithm uses a set of basis functions obtained by quantum mechanical simulation using predetermined parameters. Each basis function is optimized by subdividing it into a set of signals from magnetically equivalent spins and varying the simulated chemical shifts of each of these groups to match the signal undergoing analysis. A novel approach to the standard multidimensional minimization problem is introduced based on evaluating the fit resulting from different permutations of possible chemical shifts, obtained from one-dimensional searches. Results are presented from the analysis of (1)H proton magic angle spinning spectra of cell lines illustrating the robustness of the method in a typical application. Simulation was used to investigate the biggest peak shifts that can be tolerated.     

A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-κ (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing κ chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5–6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.     

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.     

A critical analysis of commercially available latex particle reagents for C-reactive protein (CRP) slide agglutination tests

C-reactive protein (CRP) was assessed in pediatric serum samples using different commercial latex reagents, which were analyzed for species origin of the coating antibodies, homogeneity and density of the latex particles, and prozone agglutinating capacity. All reagents correctly agglutinated the positive and negative control sera. The antibodies coating the particles differed with regard to species origin: one was coated with rabbit, one with horse and goat, one with horse, goat, rabbit and swine, while the reference reagent had horse, goat and rabbit antibodies.Only the monospecies specific antibody-coated latex showed obvious prozoning; this reagent also had the smallest and most homogenous latex particles and showed the most clear-cut reactions. False agglutination was observed at 7–26% according to quantitation with the spot immunoprecipitate assay, which compared favorably with radial immunodiffusion measurements. The lowest percentage of false readings was noted for the rabbit antibody-coated particles; the highest for the reagent with particles coated using antibodies from 4 different species.No reagent had satisfactory precision for the low positive sera between 10 and 40 mg CRP/1.     

New detection schemes in liquid chromatography

A micropolarimeter interfaced to a liquid chromatograph is shown to be suitable for selective monitoring of the optically-active components in complex samples. When an optically-active eluent is used, indirect determination of even optically-inactive materials is possible, down to the level of 10 ng of an injected component. If a second chromatogram is obtained using the racemic analogue of the optically-active eluent, quantitation can be achieved without standards and without prior analyte identification. This concept is also applicable to the refractive index detector, the absorption detector and the conductivity detector in the special case of ion chromatography, and the ultrasonic detector in gas chromatography.