Augmented mandibular bone structurally adapts to functional loading.

Long-term changes in trabecular bone structure during the 10 years following onlay grafting with simultaneous mandibular implant placement were studied. Extraoral radiographs of both mandibular sides in eight patients were taken regularly. Bone structure was analysed using a custom-written image analysis program. Parameters studied were trabecular area and perimeter and marrow cavity area and perimeter. After skeletonisation of the trabecular network, the number of end points and branching points, skeleton length, and branch angle were determined. The observed structural changes agree with the development of a more complex and more delicate or fine osseous structure. The bone shows more trabecular branching. All changes are most pronounced in the graft spongiosa, but are also found in the graft cortex and in the original mandible. The mean trabecular branch angle becomes more horizontal. The applied technique can be used to analyse long-term changes in the architecture of bone grafts. Changes found in the graft architecture correspond to changes expected after functional adaptation to loading.

Immunohistochemical expression of tenascin in melanocytic tumours of dogs.

The aim of this study was to investigate tenascin-C (TN) immunolabelling and labelling for endothelium by von Willebrand Factor (vWF) in melanocytic tumours of dogs as compared with normal tissues, to evaluate the TN distribution in these types of tumours and to investigate whether a relation could be established between TN and angiogenesis in different types of tumour. Samples of normal dog skin (n=8), benign skin melanocytomas (n=10), malignant oral melanomas (n=9) and malignant toe melanomas (n=5) were studied. The percentages of TN and vWF immunolabelling per total microscopical area were analysed by morphometric methods. In normal skin, TN was found at dermo-epidermal junctions, around hair follicles, in the smooth muscles of hair follicles, and in the walls of blood vessels. TN immunolabelling (distribution and intensity) in melanocytomas was comparable with that found in normal skin. In melanomas, TN expression was considerably increased, its intensity in toe melanomas being twice that observed in oral melanomas. The degree of TN immunolabelling was not related to the histological malignancy of the melanomas. In melanomas, TN was found in the connective tissue surrounding the tumour cell nests and in narrow stromal strands inside the tumour. Regions infiltrated with lymphocytes were devoid of TN. The presence of TN around capillaries in melanocytomas and melanomas was investigated by double-immunolabelling (for TN and vWF). The intensity of vWF and TN immunolabelling was higher in melanomas than in melanocytomas, and higher in toe melanomas than in oral melanomas; however, no clear relation between TN expression and immunolabelling for vWF was found.

Image processing and analysis program for measurement of bone density changes in reference and follow-up standardized extraoral oblique lateral cephalometric radiographs of the mandible.

OBJECTIVES: The aim of this study was to develop an image processing and analysis program for peri-implant bone density measurements of the mandible using extraoral radiographs, which includes a correction for the variable projection of the soft tissues of the face. METHODS: The measurement procedure is based on pairs of reference and follow-up extraoral oblique lateral cephalometric radiographs (OLCRs) of patients with endosseous implants in the anterior part of the atrophic edentulous mandible. The procedure consists of image acquisition, correction for radiographic variation using an aluminium wedge (i.e. film exposure and development) and transformation of the grey values into aluminium-equivalent values. After correction for variation in the projection of the soft tissues of the face using internal calibration fields, the actual peri-implant bone density measurements are performed. RESULTS: The soft tissue projection correction significantly reduces the variation between radiographs owing to the position of the soft tissues. CONCLUSIONS: It is concluded that the described image processing and analysis program, in combination with extraorally made OLCRs, is a valuable technique for measurement of peri-implant bone density changes of the mandible. With minor adaptations, the program can be used for other semi-edentulous patients.

[Onlay grafts in combination with endosseous implants in severe mandibular atrophy. A prospective radiological study]. / Botopbouwen in combinatie met implantaten bij sterke atrofie van de onderkaak. Een prospectief röntgenologisch onderzoek.

The placement of endosseous implants in combination with iliac crest onlay grafting of the anterior mandible is one of the treatment modalities for extreme atrophy of the mandible. The remodelling of these onlay grafts was studied using standardized extraoral oblique lateral cephalometric radiographs (OLCRs). A group of 8 patients was used in this prospective study. The measurements obtained from the OLCRs indicated the existence of the following stages in the process of remodelling of the bone grafts: 1. decrease in thickness and radiographic density of the (upper) cortex of the graft, predominantly during the first half year after grafting; 2. no significant changes in the radiographic density of the upper spongeous part of the graft; 3. a mean decrease of approximately 25% in the overall thickness of the graft, particularly during the first half year; and 4. an increase in the radiographic density of the lower part of the spongeous bone in the second half year after grafting. It is concluded that the remodelling of the graft has a predictable pattern in time. Densitometric measurement using standardized OLCRs can be a useful tool to evaluate quantitative changes of bone grafts to the mandible. The treatment described can be used on very strict indications only.

A novel method for the isolation of gastro-intestinal nematode eggs that allows automated analysis of digital images of egg preparations and high throughput screening.

A simple and robust method for the isolation of gastro-intestinal nematode eggs from faeces is described that uses both salt- and sugar solutions for flotation. Application of this 'salt-sugar' isolation method to large numbers of faecal samples of adult dairy cows indicates a 3- or 4-fold reduction in the proportion of e.p.g.-negative cows relative to studies that used other techniques for egg isolation. The procedure detects more eggs than the Wisconsin flotation method in replicate samples and in spiked egg-free faeces. The number of recovered eggs in spiked faecal samples is linear over a range of egg concentrations, and the transparent faecal preparations that result from the protocol can be stored as digital images which can be used as input for an efficient automated egg-counting procedure. The increased rate of processing of faeces combined with the large reduction of the percentage of e.p.g.-negative cows allows more accurate analysis of large numbers of adult or resistant animals for studies of nematode parasitism.

Oblique lateral cephalometric radiographs of the mandible in implantology: usefulness and reproducibility of the technique in quantitative densitometric measurements of the mandible in vivo.

In the literature intraoral periapical radiographs are commonly used for densitometric measurements of the mandible with implants. These films give detailed information of the implant and the surrounding bone. However, in extreme mandibular atrophy it can be difficult to obtain intraoral radiographs of adequate diagnostic quality. Extraoral Oblique Lateral Cephalometric Radiographs (OLCRs) can then be the alternative: reproducible images of large parts of the mandible can be obtained. In vitro, the results of densitometry using periapical films and OLCRs were shown to be similar. The present study aims to determine the measurement error of densitometry with OLCRs in vivo. In 16 patients (group I) with atrophic mandible and implants, duplicate OLCRs of one side of the jaw were obtained. The error of measurement for the densitometric measurements of the mandibular bone was 5.5%. The use of a specially developed correction program to compensate for undesired variations in the projection of the soft tissues of the face (tongue, lips, cheek and neck) on the radiographs resulted in a 40% reduction of that measurement error to 3.5%. This remaining error is mainly brought about by an imperfect repositioning of the patient when the duplicate OLCRs are obtained. The error caused by the image acquisition, processing and measurement is less than 1%. Deliberate variation up to 7.5 degrees of the horizontal angle wherein the OLCRs are made, results in a large error of measurement of 13.5% (group II: 17 patients). To reduce this variation the additional soft tissue correction program is unsuitable. It is concluded from this study that the described radiographic and image analysis technique is a promising tool for prospective densitometric studies of the mandible with or without implants. Especially in mandibles with bone grafts and implants, substantial changes in the graft can occur. The described technique may be particularly valuable in analyzing these changes.

Onlay grafts in combination with endosseous implants in severe mandibular atrophy: one year results of a prospective, quantitative radiological study.

In taking an overall view of the radiographs made of patients that underwent simultaneous onlay-grafting and implant placement in the anterior mandible, changes are seen in the grafts. The impression arose that a number of stages could be distinguished in this process of remodelling of the graft. The purpose of the present study was to objectivate the existence of those stages in the first year after bone-grafting by densitometric and height measurements of the onlay grafts using standardized extraoral radiographs (OLCR) and densitometric analysis in a group of 8 patients. OLCRs were taken at regular intervals in the first year after surgery. The measurements were performed in three fixed fields, both ventrally and dorsally of each implant. Generally the densitometric measurement fields were located at the (upper) cortex, the upper spongiosa and the lower spongiosa of the onlay graft. The OLCRs were also used for measurements of the height of the complete graft and of the thickness of the cortex of the graft, using a gauged caliper. The results indicate the following remodelling of the structure of the onlay grafts: 1) decrease in the thickness and the radiographic density of the (upper) cortex of the onlay graft predominantly in the first 6-month period; 2) no significant changes in the radiographic density of the upper spongeous part of the graft; 3) a decrease of approximately 25% in the overall height of the graft, particularly during the first 6-month period; 4) an increase in the radiographic density of the lower part of the spongeous bone in the second 6-month period. It is concluded that the remodelling of the graft has a predictable pattern in time: the first half year being dominated by bone resorption, mainly confined to the cortex of the graft, whereas the second half year is characterized by an increase in bone density in the (remaining) cortex as well as the lower spongeous parts of the graft.

The combined use of endosteal implants and iliac crest onlay grafts in the severely atrophic mandible: a longitudinal study.

Thirteen patients, who received an onlay bone graft augmentation to their severely atrophic mandible in combination with a simultaneous implant insertion, were studied prospectively. A reproducible method that allowed for accurate assessment of graft resorption, consisting of lateral and oblique-lateral cephalometric radiographs in combination with an image analysis system, was used to assess the resorption rate in all patients and between subgroups of patients, according to selected patient and treatment characteristics. Thirty implants were placed, none of which were lost, and all patients expressed satisfaction after a mean observation period of 877 days. A mean resorption rate of 36% of the grafted bone height occurred, mainly during the first year and with some degree of individual variance. After three years the resorption had virtually stopped. No statistically significant differences between any of the subgroups of patients could be distinguished. Peri-implantitis was found around eight implants in seven patients. It is concluded that the described surgical technique should be used only if there are stringent indications.

An image analysis system: an objective and accurate alternative for reading the agar diffusion test.

A computerized image analysis system (IAS) has been used to develop a new method for reading the agar diffusion test automatically. In four experiments a total of 88 porcine plasma and 95 urine samples were screened for oxytetracycline by the agar diffusion test. The inhibition zones were measured by hand and by the IAS directly from the bioassay plate and by the IAS from the photo-negative taken from the plate. Both methods were positively correlated with the hand method for plasma (0.9716, 0.9669) and urine (0.9878, 0.9731) in the range tested for 0.1 to 2.0 micrograms/ml. Moreover, the coefficient of variation and the day-to-day-variation amounted to 1.72% and 1.47% respectively, for the method by hand and 1.10, 1.54% and 0.27, 0.38% respectively, for the IAS methods. It is concluded that the IAS method is an objective and accurate alternative for reading the agar diffusion test.

Immunoblotting technique to study release of melanophore-stimulating hormone from individual melanotrope cells of the intermediate lobe of Xenopus laevis.

The melanotrope cells in the pars intermedia in the pituitary of Xenopus laevis synthesize and release the melanophore-stimulating hormone (alpha MSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous alpha MSH-release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit alpha MSH-release, this messenger is used to study the regulation of the heterogeneity. To quantify alpha MSH-release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small alpha MSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released alpha MSH to a nitrocellulose filter. After immunostaining, the amount of alpha MSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in alpha MSH-release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of alpha MSH-release. Dopamine treatment strongly inhibits alpha MSH-release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose-dependent as a low dopamine concentration has only a moderate effect on the alpha MSH-release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in alpha MSH-release.

Simple three-dimensional imaging of HRP-labelled neurons with the aid of an image processor.

A method is described for visualizing the three-dimensional structure of horseradish peroxidase-labelled neurons in the central nervous system of Xenopus laevis tadpoles. The reconstruction is simple, and performed on a commercially available image processing system. Minimal operational skill is required for using this method. Also, various improvements were made where user's simplicity (user-friendliness), speed and required computer memory are concerned. In addition, the excellent properties of the IBAS II system, such as image filtering and image editing functions are fully available, offering a fast, clean and complete final image reconstruction.

Flight-induced inhibition of the cerebral median peptidergic neurosecretory system in Locusta migratoria.

This study discusses the effects of a 1-hr period of flight on the peptidergic pars intercerebralis (PI)-corpus cardiacum storage part (CCS) system in male Locusta migratoria, particularly the effect on material in this system stained by a histochemical method for peptidergic neurosecretory material (NSM) or labeled by in vivo incorporation of radioactive amino acid molecules. By use of an automatic image analysis system a number of parameters of the stained or radioactively labeled substances were measured to quantify the flight-induced effects and to get information on the manner in which the neurosecretory cell bodies in the PI and their axonal endings in the CCS accommodate changing amounts of NSM. The CCS of flown locusts contained distinctly more stained and radioactively labeled substances than the CCS of unflown locusts. A tendency to similar differences was observed in the cluster of neurosecretory cell bodies in the PI. The results indicate that 1 hr flight inhibited the release of NSM by the PI-CCS system. After the onset of reduced release activity by flight, some NSM continued to be synthesized and transported from the PI to the CCS, gradually filling up and expanding the entire PI-CCS system, the NSM at the same time becoming more and more densely packed. It is concluded that the peptidergic PI-CCS system is not actively involved in the control of flight metabolism or flight behavior.

Purification of gonadotropic hormone from the pituitary of the African catfish, Clarias gariepinus (Burchell), and the development of a homologous radioimmunoassay.

Glycoprotein gonadotropic hormone (GTH) was purified from 2000 pituitaries of male and female African catfish, Clarias gariepinus. The first step was chromatography on concanavalin A-Sepharose followed by filtration on Ultrogel Aca 54, chromatography on DEAE-cellulose, and filtration on Ultrogel Aca 54, respectively. Finally, the purified fractions were analyzed by polyacrylamide gel electrophoresis. The gonadotropic activity in the different fractions was characterized using two tests: the radioimmunoassay for carp gonadotropin-beta subunit was used to quantify the immunoreactive GTH and a cAMP accumulation test was applied to measure the GTH biological activity. The purified glycoprotein GTH was used to raise antibodies and to develop a radioimmunoassay. This resulted in an assay with a variation between assays of approximately 4%, a precision of 4-8%, and an accuracy of 4-8%. GTH levels can be measured over a range of 0.8 to 12.5 ng/ml.

Topography of binding sites for neurohypophyseal hormones in rat brain.

Binding sites for arginine-vasopressin (AVP) and oxytocin (OT) were detected in neuroanatomically defined regions of rat brain by using in vitro incubation of 32 micron brain sections with the tritium-labelled ligands and autoradiography with LKB Ultrofilm. Sites were quantified by computerized densitometry of the film images. The highest amount of [3H]AVP-labelled binding sites were observed in the ventral subiculum of the hippocampus, central amygdala nucleus, lateral septum, olfactory nucleus and nucleus tractus solitarii, while the dentate gyrus contained a large amount of low affinity binding. The binding pattern of [3H]OT was more discrete. Relatively high concentrations of OT binding sites were observed in the central amygdala, ventral subiculum and olfactory nucleus. The lateral septum and the nucleus tractus solitarii were virtually devoid of displaceable OT binding while no significant association of OT with the dentate gyrus was found. The topography of binding sites for neurohypophyseal hormones corresponds to the localization of responsive sites in behavioural, biochemical and electrophysiological studies.

Catecholaminergic and serotoninergic brain structures in European green frogs: an autoradiographical study.

Aminergic brain structures have been investigated by means of light microscopical autoradiography after injection of the tritiated catecholamines noradrenaline and dopamine and the indoleamine (or tryptamine) serotonin into the brain cavity of frogs of the Rana esculenta complex. These amines are fairly specifically taken up by catecholaminergic and serotoninergic neurons, respectively, which are located in structures like the catecholaminergic preoptic recess organ; the mixed catecholaminergic-serotoninergic paraventricular organ/nucleus infundibularis-complex and nucleus reticularis mesencephali; the telencephalic septal and striatal areas and the tectum opticum, which contain many catecholaminergic axon terminals; the habenular area, which contains serotoninergic axon terminals. The autoradiographical data on the location and the nature of these aminergic brain structures agree well with the mainly fluorescence microscopical and immunocytochemical data from the literature. The autoradiographical detection method can be combined at the light and the electron microscopical level with other histological, histochemical, or immunohistochemical techniques in one and the same preparation, and the results of the different treatments may eventually be made visible simultaneously.

Arginine-vasopressin binding sites in rat brain: a quantitative autoradiographic study.

Specific binding sites for arginine-vasopressin (AVP) were detected in rat brain after incubation of tissue sections with [3H]AVP. AVP and two selective AVP antagonists are capable of displacing [3H]AVP with an IC50 in the 10(-8)-10(-7) molar range, while oxytocin and ACTH4-10 were much less effective. The neuroanatomical distribution of [3H]AVP-labeled sites was studied with autoradiography utilizing tritium-sensitive LKB film and computerized densitometry for quantitative analyses of the film images. The highest density of [3H]AVP binding sites was observed in hippocampal regions, the lateral septum, olfactory and amygdaloid nuclei, and the nucleus tractus solitarii (NTS) of the brainstem.

Accumulation of glycoprotein gonadotropin in the pituitary of juvenile rainbow trout in response to androgens and C21-steroids, including 11-steroids.

Effects of steroids on the accumulation of glycoprotein gonadotropin (GTH) in pituitaries of juvenile trout were investigated by means of scanning cytophotometry applied to immunocytochemical preparations, and with the use of a radioimmunoassay. Effects on other aspects of GTH-cell activity were analyzed by measuring the size of the gonadotrops and their nuclei. Progesterone added to aquarium water and methyltestosterone incorporated into the food showed a pronounced stimulatory effect on the accumulation of GTH. To a lesser extent, treatment with cortisol, cortisone, and desoxycorticosterone acetate administered to aquarium water, and 11 beta-hydroxy-androstenedione added to the food resulted in an increase of the hypophysial content of GTH. Steroids stimulating the accumulation of GTH in the pituitary also exhibited a positive effect on GTH-cell activity as indicated by an increase in the size of gonadotropic cells. Progesterone incorporated into the food did not influence the GTH-content and the GTH-cell activity. It is suggested that the route of administration of an exogenous steroid is essential for its effect on GTH cells in trout. Comparison of GTH values reveals an excellent correlation between the data from the radioimmunoassay and those from the corresponding densitometric measurements. No correlation was observed between values of morphometrically determined GTH-cell activity and the densitometric values reflecting hypophysial GTH content.

Quantitative estimation of pituitary gonadotropin in juvenile rainbow trout with scanning cytophotometry applied to immunocytochemical preparations.

A densitometrical method with computer-controlled scanning cytophotometry has been developed to quantify the amount of gonadotropin (GTH) in immunocytochemically treated paraffin sections of pituitaries of juvenile rainbow trout. Results obtained with this method totally correspond with those established with a radioimmunoassay of pooled pituitaries. Scanningcytophotometry offers the advantage that GTH-content of individual pituitaries of juvenile or small adult fish can be investigated.

Immunocytochemical evidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri).

In the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method. Experiments were carried out on glutaraldehyde-picric acid-acetic acid-fixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody. Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion. In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.