C. Vyvyan Howard

Estimating the length of a bounded curve in three dimensions using total vertical projections

A new method is proposed to estimate the total length of a bounded, isolated linear feature in three dimensions from ‘total vertical projections’, obtained by rotating the curve about a fixed axis (arbitrarily called ‘vertical’) and projecting it onto a fixed vertical plane. No sections are required. Properly stained and embedded neuron dendrites, mycelial trees, fluorescent cytoskeletal filaments within a cell, etc., are candidate specimens for the method, especially in combination with the new devices for non‐invasive three‐dimensional microscopy. It is necessary that the specimen curve is rigid (i.e. of constant shape), that its length density is not too high (so that overlapping effects are not important) and that the embedding medium is fairly transparent. Given these requirements, the method can be very accurate and convenient to use, as is exemplified here.

Renal Developmental Arrest in Sudden Infant Death Syndrome

Investigations linking sudden infant death syndrome (SIDS) and type II intrauterine growth retardation (IUGR) have thus far failed due in part to technical limitations. Recently developed stereological methods for the unbiased estimation of total nephron number in the human kidney are capable of detecting deviations from normal values of greater than 10%. We compared the total number of nephrons in the kidneys of 24 SIDS victims with those from 16 controls with the same age range. Mean nephron number was significantly (P < 0.001) reduced in ex-IUGR SIDS cases (birthweight under the 10th centile, n = 9, mean number 635,000, range 327,000-1,010,000) in comparison with controls (903,000, 740,000-1,060,000). A similarly significant (P < 0.01) reduction in the normal birthweight SIDS group (birthweight over 10th centile, n = 15, 690,000, 361,000-1,040,000) was found. This hitherto unreported renal developmental arrest may be only one manifestation of a general, somatic developmental defect, reflecting adverse intrauterine conditions; other organ systems, similarly critical to homeostasis may be comparably affected. The findings, although not proposed as direct cause of SIDS, may represent a potential explanation for the recognized association of IUGR and SIDS, and provide--we believe--the first quantitative evidence of intrauterine growth retardation in, at least a number of, children of average birthweight.

Estimating neuron dendritic length in 3D from total vertical projections and from vertical slices.

The recently developed method of total vertical projections is illustrated to estimate the total dendritic length of a human Substantia Nigra neuron. Next, the length of the different orders of dendritic branches, and the mean segment length for each order ‐ commonly regarded as important parameters in neuron physiology ‐ are also estimated. Finally, it is shown how to estimate the mean dendritic length in a population of neurons from vertical slices of arbitrary and unknown thickness. Being unbiased and highly efficient, the proposed methods offer interesting alternatives to current procedures used for the metric analysis of neuron arborizations.

Measuring the surface area of a cell by the method of the spatial grid with a CSLM—a demonstration

The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.

The application of total vertical projections for the unbiased estimation of the length of blood vessels and other structures by magnetic resonance imaging

A new stereological method has recently been developed to estimate the total length of a bounded curve in 3D from a sample of projections about a vertical axis. Unlike other methods based on serial section reconstructions, the new method is unbiased (i.e., it has zero systematic error). A basic requirement, not difficult to fulfill in many cases, is that the masking of one structure by another is not appreciable. The application of the new method to real curvilinear structures using a clinical magnetic resonance (MR) imager is illustrated. The first structure measured was a twisted water-filled glass tube of known length. The accuracy of the method was assessed: With six vertical projections, the tube length was measured to within 2% of the true value. The second example was a living bonsai tree, and the third was a clinical application of MR angiography. The possibility of applying the method to other scientific disciplines, for example, the monitoring of plant root growth, is discussed.

Environmental influences in cancer aetiology

Purpose. The purpose of this review is to inform both scientists and clinicians about the increase in cancer incidence throughout the Western World and to discuss environmental influences in cancer aetiology, in order to stimulate thoughts about plausible aetiological mechanisms and possible preventative measures.Design. Literature review.Materials and methods. This review was conducted by searching biomedical databases such as PubMed and Medline. Further research to obtain cancer incidence data involved accessing UK cancer registries, major cancer charities and government statistical records from the Office of National Statistics, the Department of Health, and the Department of Environment, Food and Rural Affairs.Results. Cancer incidence rates have increased in the Western World and this increased incidence affects the whole age spectrum. Epidemiological studies have provided some evidence of an association between exposure to environmental contaminants such as organochlorines and increased cancer risk....

Renal developmental delay expressed by reduced glomerular number and its association with growth retardation in victims of sudden infant death syndrome and in "normal" infants.

In victims of sudden infant death syndrome (SIDS), renal development has been reported to be significantly impaired. In the present study, we used stereological techniques to estimate volume of kidney cortex and total number of glomeruli in a group of human infants. Infants were classified according to cause of death—SIDS or non-SIDS. Cases were further subdivided according to birth weight—normal birth weight (NBW) or low birth weight (LBW) (we were unable to identify any non-SIDS LBW infants for our study). No significant differences were found between NBW and LBW infants (irrespective of cause of death) for cortical volume, glomerular density, or total glomerular number (p > 0.140). Kidney cortical volume, glomerular density, and total glomerular number were not significantly different between SIDS and non-SIDS infants (p > 0.510). Glomerular number was only significantly less in SIDS infants of LBW (p = 0.032) than in controls according to the Wilcoxon rank sum test; using the Kruskal-Wallis for one-way analysis, no significant difference was found (p > 0.010). These results contrast with those from previous studies, as a reduction in glomerular number was not noted in SIDS NBW infants, and the mean value for the control (non-SIDS NBW) group was significantly reduced (p < 0.01) from those of previous studies. This indicates that glomerular number reduction is seen in SIDS NBW and non-SIDS NBW cases and is therefore directly associated with growth retardation rather than with SIDS.

Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy

The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.

QUANTITATIVE MICROSCOPY IN STUDIES OF INTRAUTERINE GROWTH RETARDATION

In the past, the detection of fetal damage has tended to be restricted to the naked eye identification of major malformations, with the period of organ maturation being relatively neglected. Increasingly, however, unbiased design‐based stereology is being used in developmental toxicological studies. In the field of intrauterine growth retardation, such methods are capable of providing new insights into fetal vulnerability during critical periods in organogenesis, with consequences for both post‐natal and adult disease.

Quantitative phenotyping as an efficient means to estimate C-cell number in a knock-in mouse model of MEN2B.

Over the last two decades we have witnessed the generation of hundreds, if not thousands, of lines of genetically altered mice, large numbers of which are being produced in order to model human disease. Given that their creation is still rather technically demanding and labour intensive, the time taken analysing the resultant phenotypes should be such that the maximal amount of information can be gleaned efficiently in an unbiased manner so as to be as close to the “true” value as possible. In an attempt to characterise a cell-specific phenotype in a genetically defined knock-in mouse model of multiple endocrine neoplasia type 2B (MEN2B) we used a modern, unbiased, stereological approach called the optical fractionator to estimate total cell number in 3-D space. By applying a sampling technique to tissue blocks in a systematic random uniform manner, we demonstrate that the total number of calcitonin-immunoreactive C-cells in the thyroid glands of littermate mice harbouring activating mutations in one or both alleles of ret does not vary significantly (p= 0.46) from an unbiased estimate of 23,000 in wildtype controls; likewise, neither does mean thyroid volume (p= 0.78) when estimated using Cavalieris principle. We demonstrate that the variation associated with the quantitative phenotyping method is negligible. Using this efficient, unbiased stereological method our results provide new insights into cell number and positioning with consequences for both normal and disease states. In summary, this unbiased stereological technique is conceptually simple, can be applied efficiently, and is pertinent to quantitating a wide variety of cell phenotypes thereby bridging specialisation boundaries. We propose the adoption of this technique to mouse experimental geneticists and recommend its horizontal transmission across all fields within experimental biology.