Comparative study between biochemical and histological methods and image analysis in liver iron overload.

Iron overload has been measured in 100 hepatic biopsies by three different methods: (i) biochemical assay of the liver iron concentration (LIC), (ii) histological grading (HISTO) and (iii) automated image analysis with a Leitz Texture Analysis System by estimating two parameters, (a) the total iron area (TIA) and (b) the first grey level step (FGLS) at which the iron is detected. Image analysis appears as a specific, sensitive, quick, reproducible and valid method.

A morphometric study of mouse trigeminal ganglion after unilateral destruction of vibrissae follicles at birth.

A morphometric study of the trigeminal ganglion after unilateral vibrissae follicles' coagulation in newborn mice has shown the following: (a) a 42.8% decrease of the total volume of the ganglion on the deafferented side with reference to the normal side; a 61.5% decrease of the ophthalmic-maxillary part of the ganglion where neurons whose axons innervate vibrissae follicles are located, and only a 24.1% decrease in the common part; (b) a 54.8% decrease of the neuronal cell body volume in the ophthalmic-maxillary part and practically no change in the common part, and (c) a 64.5% decrease of the volume occupied by the nerve fibers in the ophthalmic-maxillary part and only a 28.1% decrease in the common part. A comparison of the section areas in ganglion and of the bulk area of neuronal cell bodies at different levels has also been performed. Counting of the neuronal cell bodies in the ophthalmic-maxillary part of the ganglion indicated a mean neuronal loss of 36.5%. Peripheral reinnervation of the common fur by regenerated axons of neurons which previously innervated vibrissae, although unlikely, cannot be completely excluded.

A morphometric study of mouse trigeminal roots after unilateral destruction of vibrissae follicles at birth.

Trigeminal sensory roots were studied in neonatal mice. On the deafferented side, the surface area of the cross-section through the sensory root is diminished by 31% and the number of myelinated fibers is reduced by 21%, but the proportion between myelinated and unmyelinated fibers remains unchanged. The distribution of axonal diameters, analysed in 7 dorso-ventral scanning bands through the sensory roots, indicates a loss or eventually an atrophy of large myelinated axons in the medial two thirds of the sensory root. In both control and deafferented sides the diameter of the myelinated fiber (outside the myelin sheath) is proportional to the axon diameter (inside the myelin sheath). Our results confirm the loss of most of the neurons innervating vibrissae and the lack of regeneration or sprouting in the deafferented root in the newborn mouse.

Effects of occupational lead exposure on motor and somatosensory evoked potentials.

In order to objectivate the effects of inorganic lead compounds on the nervous system, 17 men occupationally exposed have been recorded for Motor Electric Potentials (MEPs) of the abductor policis brevis muscles with transcranial, cervical root and peripheral electrical stimulations, for Somatosensory Evoked Potentials (SEPs) of median and tibial posterior nerves, and for Electromyography (EMG). Considering each subject, 16 out of the 17 lead exposed ones exhibited electrophysiological abnormalities, occurring at both peripheral and central levels for the somatosensory as well as for the motor systems. As compared to a normal control group, the lead exposed one showed decreased sensory and motor peripheral conduction velocities, increased motor central conduction time, and delayed cortical P22 SEPs component that could be interpreted as a sensory-motor cortical dysfunction. The respective contributions of MEPs and SEPs are compared to clinical, biological and EMG examinations in the evaluation of the neurotoxic effects of inorganic lead compounds.

Karyometry of pseudostratified, metaplastic and dysplastic nasal epithelium by morphometry and stereology. 1. A general model for automated image analysis of epithelia.

Nuclear enlargement, usually due to the existence of abnormally high DNA (ploidy) values, is one of the alterations most often encountered in pathological epithelia, especially in cancerous and precancerous states. Direct DNA measurements on microscopic sections are impossible, and karyometric studies must take the difficult stereologic problem of spheroid sectioning into account. A mathematical model is proposed to describe an aggregate of separate parallel anisotropic prolate spheroids of variable size and eccentricity. This model appears suitable for the study of epithelial nuclei. After a tentative estimation of the biopsy angle, the application of the model to nuclear profile measurements produces the stereologic data of the mean spheroid nucleus: major and minor axes (and their ratio), volume, surface area (and the V/S ratio), and numerical density. Considerable time may be saved by the use of an image analyser with a specific algorithm. Most of the steps are entirely automated, but the user retains the interactive choice of the nuclei to be studied, for instance by separating the different epithelial layers. The profile data are computer-filed and several programs can yield the stereologic estimations and plots of nuclear profile areas. Poolings of cases of similar histologic staging and nearly the same sectioning angle can be used for the profile area plots. The model has been applied to pseudostratified and altered nasal mucosa of nickel workers. The stereologic estimates, mainly the nuclear volume, correlate well with histologic gradings. The nuclear profile area plots are considered useful for an assessment of the nuclear polyploidism. The complete model should be applicable to many other types of functionally or pathologically altered epithelia, particularly for cancer and precancer screenings, when the use of sectioned material is a prerequisite.

[Evoked potentials and age: different aging by sex?]. / Potentiels évoqués et âge: vieillissement différential selon le sexe?

Brainstem Auditory Evoked Responses, Pattern-Shift Visual Evoked Potentials, and Short-latency Somatosensory Evoked Potentials for upper and lower limbs have been recorded in 60 male and female normal control subjects, aged 20-84 years. Age and gender effects were analyzed from the comparison of linear regressions with age and from the comparison of the mean values calculated in 3 age groups (20-40 years, 40-60 years, over 60 years). No noticeable effect of age was observed on Brainstem Auditory Evoked Responses. A gender-differential aging process is discussed for some of the short-latency Somatosensory Evoked Potentials. The constitution of reference norms which differ for male and female subjects is considered to be necessary.

[Somatosensory evoked potentials during spinal orthopedic surgery in adult patients]. / Potentiels évoqués somesthésiques au cours des interventions orthopédiques sur le rachis de l'adulte.

Somatosensory evoked potentials (SEPs) were recorded in 165 patients from 185 operations for scoliosis and major spinal distortions, lumbar or cervical stenosis, post-traumatic malunions, or tumors. They were obtained by percutaneous stimulation of right and left median and tibial posterior nerves, and recorded on the scalp as recently, motor potentials (MPs) have also been obtained, by a trans-spinous stimulation rostral to the level of intervention. The protocol consists of pre- and post-operative recordings, as well as a continuous intra-operative monitoring. Neuromonitoring is justified in operations implying neurological risks, particularly scoliosis. A durable disappearance of SEPs and MPs was always associated with post-operative neurological disorders. Transient disappearances, not associated with neurological disorders, have been observed during certain manoeuvres such as vertebral traction, osteosynthesis, placing trans-pedicular screws or hooks, leading the surgeon to modify the operation. The role of medullary ischemia on the degradation of electrophysiological signals, obvious in cardiovascular decompensations, seems predominant in the post-operative occurrence of neurological impairment. During the post-operative period, SEPs allow one to discriminate between organic disorders and subjective complaints; they help in diagnosing hematomas, and distinguish between radicular and spinal cord injuries.

Evoked potentials in workers occupationally exposed to organic solvents.

Somatosensory, Brainstem Auditory and Pattern Reversal Evoked Potentials (SEPs, BAEPs, PREPs) are recorded in workers occupationally exposed to mixtures of organic solvents, in order to specify the levels of the nervous system affected by a long term exposure to solvents, and to analyze the effects of age and gender. The most significant differences are found for SEPs; they objectivate peripheral impairments magnifying the differential effects of age observed in the control subjects, and show the higher sensitivity of women. The central impairment is pointed out by the latency delay of P22 component mainly, whose age-related increase is amplified by solvent exposure.

Measurement of spermatozoal motility over prolonged periods of time using image analysis.

This paper presents a new method for the quantitative analysis of the movement characteristics of spermatozoa. The method is based on (i) measuring the displacement of a spermatozoon observed using a microscope and (ii) automatically compensating for the displacement of the spermatozoon in order to keep it in the center of the microscopic field. The displacement of the spermatozoon is measured by on-line processing of digitized images of the microscopic field, acquired by an image analysis system. The motility characteristics of the same spermatozoon can be evaluated over prolonged periods of time (typically 10 min). The movement characteristics of 19 spermatozoa from five fertile donors were studied. At constant temperature, intradonor variations of the motility parameters were found. No statistically significant differences were found in the mean values of the motility characteristics for three of four donors. At increasing temperature, the mean curvilinear velocity, averaged on the 19 spermatozoa from the five donors, regularly increased between 23 and 30 degrees C and remained constant up to 38 degrees C.

Postnatal maturation of the vascularisation of the suprasylvian gyrus of the cat.

Vascular growth in the median suprasylvian gyrus of the cat has been analysed quantitatively with respect to mitoses, vascular density, vascular diameters, vascular coefficient and preferential vascular orientation. After correction for shrinkage and growth, four maturation periods were identified: (i) Immature period (first postnatal week), when the tissue exhibited numerous vascular mitoses, a low but constant vascular density and vascular coefficient, preferentially radiate vessels and immature (small) vascular diameters. (ii) Premature period (second to fourth week), with few mitoses, a rapidly increasing vascular density, immature (small) vascular diameters, an increase in the number of vessels more than 10 microns in diameter, and a preferential orientation of tangential vessels. (iii) Pre-adult period (fifth to sixth week), without mitoses. The vascular density increased greatly, the vascular diameters reached adult profile, the number of vessels over 10 microns in diameter decreased, the vascular orientations were both tangential and radiate and the vascular coefficient remained slight. (iv) Adult period, when the vascular density and the vascular coefficient were maximal, the vessel diameters were of adult type, and the orientation was tangential. From six weeks, the vessels increased only in length. Vascular maturation proceeded from the depth toward the surface of the cerebral cortex. Layers II-IV were those most highly vascularised, regardless of age. This is discussed in relation to synaptic growth. There was no direct relation between vasculogenesis and myelination. The white matter had a typically radiate vascularisation. A transitional zone between cortex and white matter was identified. It had a loose mesh vascular network and corresponded to the area in which dendrites of inverted pyramidal cells were found. The role of immature vascularisation in the nutrition of the neuropil is discussed.

Software package for the quantitative image analysis of histological sections.

A software package of general interest, and adapted to the analysis of histological sections has been written. The different programs of this package have been classified into four groups: macroanalysis, selection of fields, analysis, and data processing. The macroanalysis allows the acquisition of topographical information that will be used during the selection of fields and the analysis. Mapping programs have been added to the classical data processing of the measured parameters (statistical calculations, edition of values, histograms, graphs, .. .). They allow the generation of parametric images representing the distribution of particular elements in the section, or the topographical variations of calculated parameters. The recombination of information obtained at different magnifications, and the correlation of information of different types is thus possible and of great interest in the study of complex histological structures.

An image analysis morphometric method for the study of myelinated nerve fibers from mouse trigeminal root.

For the morphometric light microscopic study of myelinated fibers in mouse trigeminal root, it was necessary to write: (1) an entirely automatic analysis program for the myelinated axons inside the myelin sheath, based on the detection of the myelin sheaths, and (2) an interactive analysis program for the myelinated fibers outside the myelin sheath, due to the high density of compactness of the myelinated fibers based on an indirect fiber individualization by reconstructing them from their axons. In the latter, a semiautomatic correction method (drawing the profile contours with a light pen) allowed compensation for the failures of the automatic method, except for the smallest fibers, which represented 8% of the total. Using these programs, 95% of the axons could be measured and 92% of the myelinated fibers whose axons were analyzed could be measured. The area-equivalent diameter was independent of the detection method; it is a correct-size measurement parameter for axons and fibers that is unrelated to their shape. The projected diameter, an estimation of the perimeter obtained by measurement of the profile projections, depended upon the detection method because the profile contour was influenced by the detection method; it thus takes into account the profile shape. For myelinated fibers, whose analysis program used two detection methods (automatic and semiautomatic), there was an average difference of 16% between the projected diameters obtained with these two methods, whereas the equivalent diameter value was the same. The fiber circularity factor could not be precisely estimated because of the detection error; the axon circularity factor was more reliable since the axon detection was completely automatic.

Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures.

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.

Development of immunologically identified brain cells in culture: quantitative aspects.

Mechanically dissociated brain cells of 14 and 18-day-old mouse embryos and of mouse neonates were cultured for 3 weeks. Neurons, oligodendrocytes and astrocytes were identified at the 7th, 14th and 21st day in vitro by staining the cultures using the indirect immunoperoxidase technique with antisera directed against neuron specific enolase, galactocerebroside, myelin basic protein and glial fibrillary acidic protein. The number of neurons and oligodendrocytes was higher in embryonic cultures than in neonate cultures. The expression of some antigens was also different in the two types of culture. Our results indicate that the development of brain cells in mechanically dissociated brain cell cultures depends on the age of the animal at the time of plating.