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PubMedDate not supplied

Dissection autoradiography: a screening technique using storage phosphor autoradiography to detect the biodistribution of radiolabelled compounds.

INTRODUCTION: This study reports an alternative, rapid, whole body autoradiography technique which utilises storage-phosphor imaging technology. Conventionally, tissue or whole body sections have been used to examine the distribution of radiolabelled test compounds. However, the information acquired relates only to the sections examined, and the amount of radioactivity within the whole organ cannot be quantified. We have developed a rapid semi-quantitative technique that produces a concise visual representation of the distribution of the isotope throughout the entire animal: dissection autoradiography (DAR). METHODS: By dissecting a mouse which has been administered 14C-labelled methotrexate (MTX) and drying the tissues on a gel dryer, whole organs and aliquots of body fluids can be exposed to a phosphor imaging plate. The data obtained was analysed with the software associated with the phosphor imaging system and, by using 14C standards, the amount of 14C per total organ or tissue was quantified relative to other samples. Another widely used method to detect radiolabelled material in vivo is tissue solubilisation (TS) followed by liquid scintillation counting (LSC). This conventional method was compared with DAR. RESULTS: The new technique described in this communication was found to have a high level of reproducibility (R(2)= 88-95%). Whilst DAR was less sensitive than TS and LSC, trends over time in the biodistribution of 14C-MTX throughout most tissues were consistent between techniques. DISCUSSION: Whilst TS and LSC was a more sensitive technique, it was labour intensive and expensive in terms of consumables and time when compared with DAR. Dissection autoradiography has the potential to be used to screen quickly large numbers of samples in the biodistribution studies of various conjugates, isomers, derivatives or formulations of a parent compound, following a variety of routes of administration.

Animals↗
PubMed1987

Brainstem projections to lumbar motoneurons in rat--I. An ultrastructural study using autoradiography and the combination of autoradiography and horseradish peroxidase histochemistry.

In 11 rats the descending projections from the ventrolateral medullary medial reticular formation, the medullary raphe nuclei and the area of the nucleus coeruleus and subcoeruleus to lumbar motoneuronal cell groups were studied by means of electron microscopical autoradiography after [3H]leucine injections in the respective brainstem areas. The distribution of the transported radioactivity in the autoradiographs was determined using the circle method [Williams (1977), in Practical Methods in Electron Microscopy, Vol. 6, pp. 85-173] which showed that the vast majority of the silver grains was located over terminal profiles. In the motoneuronal cell groups six different types of terminals were distinguished. After injections in the ventrolateral medial reticular formation the majority of the silver grains was located over F-type terminal profiles while many fewer silver grains were found over S- and G-types. After injections in the raphe nuclei and the adjoining medial reticular formation approximately equal numbers of silver grains were found over F- and G-type terminals while fewer were found over S-type. A small proportion of silver grains was present over C-type terminals and only after injections in the ventrolateral medial reticular formation. After [3H]leucine injections in the area of the nucleus coeruleus and subcoeruleus the majority of silver grains were located over E- and S-type terminals whereas relatively few were located over F-type terminals. The E-type terminal, which has not been described before in the motoneuronal cell groups, is characterized by the fact that it contains relatively small vesicles and occasionally elongated or canaliculi-like structures. In the three groups of experiments approximately 40-50% of the labelled S- and F-type terminal profiles established synaptic contacts, but only approximately 10% of the labelled E- and G-type terminal profiles did so. In all cases these synaptic contacts were established mainly with proximal dendrites. In the autoradiographs some of the silver grains were concentrated into clusters. The vast majority of these clusters, consisting of six or more silver grains, were centred over terminal profiles. The differential distribution of these clusters over the different types of terminal profiles in the various experiments was roughly the same as found by means of the circle method. In two rats [3H]leucine injections in the ventrolateral medial reticular formation were combined with horseradish peroxidase injections in the ipsilateral hindleg muscles, resulting in retrograde labelling of the corresponding motoneurons as visualized by means of the tetramethyl benzidine incubation method.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Studies on the quantitative autoradiography. III. Quantitative comparison of a novel tissue-mold measurement technique "paste-mold method," to the semiquantitative whole body autoradiography (WBA), using the same animals.

A novel preparation technique, so called "Paste Mold," was devised for organ and tissue distribution studies. This is the most powerful by joining with autoradioluminography (ARLG), which was established and validated recently in the working group of Forum '93 of Japanese Society for study of xenobiotics. A small piece (10-50 mg) of each organ or tissue was available for measuring its radioactive concentration and it was sampled from the remains of frozen carcass used for macroautoradiography (MARG). The solubilization of the frozen pieces was performed with mixing a suitable volume of gelatine and strong alkaline solution prior to mild heating kept at 40 degrees C for a few hours. After that, the tissue paste was molded in template pattern to form the small plates. The molded plates were contacted with Imaging plate (IP) for recording their radioactive concentration. The recorded IP was processed by BAS2000. The molded plate was formed in thickness of 200 microns, so called infinit thickness against soft beta rays, and therefore the resulting relative intensities, represented by (PSL-BG)/S values, indicated practically responsible ratio of the radioactive concentration in organs and tissues, without any calibulation for beta-self absorption coefficiency. On the other hand, the left half body of the frozen carcass was used for making whole body autoradiography (WBA) before the Paste-Mold preparation. Comparison was performed for difference in (PSL-BG)/S values of organs and tissues between frozen and dried sections. A good concordance in relative intensities, (PSL-BG)/S by the Paste-Mold preparation was given with those by the frozen sections rather than dried sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Studies on the quantitative autoradiography. II. Radioluminography for quantitative autoradiography of 3H.

The process of obtaining 3H-autoradiographic (ARG) images has been expected for a long time. A few X-ray films with no protection layer are commercially available, however, they do not give a reliable image which can be quantitated and can give good contrast. We tried to fabricate a 3H-type sensor which has no protection layer on a highly sensitive sensor, and called it Imaging plate (IP). The IP is composed of one of the specially designed photo-stimulable phosphors containing of BaFX: Eu2+(X = Cl, Br or I) crystals. Results indicated a good contrast and reliable whole-body ARG image of 3H-labelled glucose with trial IP, which has never been obtained with any X-ray films even if these were subjected to a long exposure time. The ARG image can be displayed by either black-and-white hard copy or a colored one with the digital display representing relative intensity of photostimulated luminescence (PSL) without back ground (BG) or relative intensity concentration ((PSL-BG)/S), where S was equivalent to 100 pixels (1 mm2). Similarly to the experimental results of 14C, the linear relationship relative intensity and radioactivity of the 3H standard sources was demonstrated with a very wide range of 10(2) to 10(5) dpm/mg upon the exposure for 7d. Also the relationship between relative intensity and relative exposure (radioactivity x exposure time) was linear within the latitude of relative intensity 10(1) to 10(5) (PSL-BG). The trial IP was particularly effective for the quantitative autoradiography of TLC plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1979

Whole-body autoradiography of bumetanide-14C in dogs by round saw method.--A combined method of autoradiography and tissue radioassay--.

The distribution of a new potent diuretic, Bumetanide, in dogs was studied by means of a combined method of whole-body autoradiography and tissue radioassay, after oral administration of Bumetanide-14C to five young beagle dogs (ca. 1.2 kg). A round saw method developed by Kalberer was applied to obtain frozen slices of dog whole-body with a thickness of 3 mm. After exposure to X-ray films from both sides of the slices at -60 degrees C, frozen samples of the main tissues as well as the blood, urine, bile and intestinal contents were obtained to be assayed for radioactivity and to be analyzed for metabolites by TLC. The advantages of the round saw method (thick slices) in larger animals as dogs and monkeys as compared to the conventional microtome method (thin sections) were pointed out and discussed.

Administration, Oral↗
PubMed1984

Stimulation of wound healing, using brain extract with fibroblast growth factor (FGF) activity. III. Electron microscopy, autoradiography, and ultrastructural autoradiography of granulation tissue.

A bovine brain fraction with FGF activity (fibroblast growth factor) increased the formation of granulation tissue of rats in an experimental wound healing model. It was characterised by intensified formation of new capillaries, stimulation of the synthesis function of fibroblasts and myofibroblasts, and proliferation of freshly developed granulation tissue. The results obtained from light microscopy and autoradiographic electron microscopy with 3H-thymidine suggested significant increase in the labelling of activated, undifferentiated endothelial cells in vascular sprouts and of differentiated endothelial cells in developed capillaries. FGF S3 appeared to speed up the differentiation of freshly formed endothelial cells. Also labelled were pericytes and other fibroblastoid cells, undifferentiated mesenchymal cells, juvenile undifferentiated fibroblasts, mature fibroblasts, myofibroblasts, and macrophages. Pericytes are presumed to be preferential precursors of myofibroblasts, on account of their occurrence in time and similar ultrastructural parameters. Close neighbourhood of lymphocytes and labelled fibroblasts gave rise to the assumption of specific effects, such as stimulation by lymphokines.

Animals↗
PubMed1970

Nonphotographic alpha autoradiography and neutron-induced autoradiography.

With a new combination of two techniques, (i) alpha-particle or fission fragment registration in thin polymer foils by etching and (ii) automatic counting and magnification of the etched perforations by local evaporation of a thin metal layer with an electric spark, the sensitivity of conventional photographic methods for determining quantity and spatial distribution of alpha emitters, fissile materials, and of elements undergoing (n, alpha) reactions can be drastically improved, without need for darkroom processing and microscopic evaluation.

Alpha Particles↗
PubMed1995

Studies on the quantitative autoradiography. I. Radioluminography for quantitative autoradiography of 14C.

Evidence is presented to elucidate whether or not quantitative analysis of autoradiographs can be obtained with a 14C-labelled compound by the use of a new type of radiosensor called Imaging plate (IP). Our results showed that a linear relationship between relative intensity of PSL-BG and radioactivity of a given area was obtained. The linearity was maintained in a relatively wide range from 10(1) to 10(5) dpm of radioactivity. About 100 times higher sensitivity of the IP than any commercially available X-ray film was demonstrated for not only 14C-radioactive standard sources but also experimentally obtained 14C-radioactive spots developed on a TLC plate. Results also showed a linear relationship between relative intensity and radioactivity of the specimens in a very wide range from 10(1) to 10(5) dpm/mg after exposure for 7d. Furthermore, the relationship between PSL-BG and relative exposure (radioactivity x exposure time) was linear when PSL-BG was from 10(1) to 10(5). In addition to the above properties, a combination of IP and BAS2000, a computerized image display system, was able to completely erase the background prior to use.

Animals↗
PubMed2001

An overview on functional receptor autoradiography using [35S]GTPgammaS.

[35S]GTPgammaS binding autoradiography is a novel method to study the distribution and function of neurotransmitter receptors in tissue sections. This technique unifies the advantages of receptor-autoradiography and [35S]GTPgammaS binding, providing anatomical and functional information at the same time. Due to these two main features, it can also be called 'functional autoradiography'. [35S]GTPgammaS binding has long been used to study the first step of the intracellular signaling pathway, but until the mid 1990s it has only been performed on cell membrane extracts. Functional autoradiography evolved from this biochemical assay and ligand autoradiography, and is based on the increase in guanine nucleotide exchange at G-proteins upon agonist stimulation. With the technique, activation of G-protein-coupled receptors upon agonist binding can be detected, and, at the same time, the location of activated receptors can also be visualized. Thus only those presumably active G-protein-coupled receptors are visualized that can be involved in signal transduction. In the past 5 years the technique has become more and more frequently used in neuroscience, and it has been adapted to several receptors in different species, including also the human brain. [35S]GTPgammaS binding autoradiography can be used to describe the distribution of G-protein-coupled receptors. Some inferences on their coupling efficiency can also be drawn. Besides the localization of ligand binding sites, it provides information on the action of the ligand on the receptor: agonists, antagonists, and inverse agonists can clearly be distinguished. Moreover, [35S]GTPgammaS binding autoradiography can successfully be combined with other in vitro assays, like receptor autoradiography, in situ hybridization histochemistry, or even with biochemical and electrophysiological experiments. This review presents an overview on the history and the development of this technique. Its main advantages and limitations are summarized, together with a few basic technical questions. A number of experiments performed with [35S]GTPgammaS binding autoradiography so far, and some possible applications for the future, are also reviewed.

Animals↗
PubMed1995

Localization and quantification of cholecystokinin receptors in rat brain with storage phosphor autoradiography.

The present study evaluated a new imaging technique that demonstrated the application of storage phosphor autoradiography in the localization and quantification of cholecystokinin receptors in rat brains and compared the results with film autoradiography. Cryostat sections were incubated with [125I]Bolton-Hunter-labeled sulfated cholecystokinin octapeptide followed by exposure to a storage phosphor-imaging screen and suitable autoradiography film. To obtain satisfactory images, it took 6 days with film autoradiography vs. 15 hours with the storage phosphor technique. Both film and storage phosphor autoradiograms showed the same cholecystokinin receptor distribution in brain sections; however, the film imaged more details. To reach the lowest possible response ratio between low and high receptor density regions in rat brains, storage phosphor autoradiography was about 240-fold faster than film. In addition, the new technique presented a considerably larger exposure time range for maintaining that ratio. The binding per area showed a linear relationship with the thickness of sections between 5 and 14 microns. In the linear response range, the quantitative results of both methods are comparable. In conclusion, storage phosphor autoradiography is a faster technique for localizing and quantifying peptide receptors in tissue sections but slightly compromised in resolution when compared with film autoradiography.

Animals↗
PubMed1989

Neuroanatomical localization of sex steroid-concentrating cells in the Japanese quail (Coturnix japonica): autoradiography with [3H]-testosterone, [3H]-estradiol, and [3H]-dihydrotestosterone.

Steroid autoradiography was undertaken to determine the neuroanatomical loci which might be involved in the activation of steroid-sensitive behaviors in the Japanese quail (Coturnix japonica). Male and female quail were either surgically gonadectomized or photically regressed and implanted with androgen or estrogen to restore normal sexual and courtship behavior. After gonadectomy or implant removal, each quail was injected with 250 microCi of [3H]-testosterone (3H-T), [3H]-estradiol (3H-E2), or [3H]-dihydrotestosterone (3H-DHT), sacrificed, processed for autoradiography, and the telencephalon, diencephalon, mesencephalon, and rhombencephalon were examined for labelled cells. Following 3H-T or 3H-E2 injection and autoradiography, labelled cells were found in nucleus septalis lateralis (SL), nucleus preopticus medialis (POM), nucleus paraventricularis (PVN), regio lateralis hypothalami (LHy), nucleus inferior hypothalami (IH), nucleus infundibuli (IN), nucleus intercollicularis (ICo), substantia grisea centralis (GCt), nucleus taeniae (Tn), and in the reticular formation near nucleus motorius nervi trigemini (MV). In addition, following 3H-E2 autoradiography, labelled cells were found around nucleus accumbens (Ac). Following 3H-DHT autoradiography, labelled cells were found only in SL, PVN, Tn, LHy, ICo, and CGt. No labelled cells were found in Ac, POM, IH, IN, or MV even after long exposure times. These results suggest that the nuclei labelled following 3H-E2 but not 3H-DHT administration bind exclusively the aromatized metabolites of T. Since quail show a sex difference in male-typical copulatory behavior in response to E2, labelled cells were counted in POM, LHy, IH, and Tn of male and female quail following 3H-E2 injection and autoradiography. No sex differences in the number of labelled cells were found in POM, LHy, or IH. Males were found to have more labelled cells than females in Tn. These results show that sex differences in male-typical copulatory behavior are not due to sex differences in the number of cells binding estrogens in POM. The results reported here constitute the most neuroanatomically extensive report of steroid binding cells to date for a galliform brain, the first comparison in a galliform bird of the distributions of cells labelled following injection of 3H-T, 3H-E2, and 3H-DHT and the first analysis of sex differences in numbers of estrogen-binding cells in four nuclei in the avian brain.

Animals↗
PubMed2005

Value of immunohistochemistry for somatostatin receptor subtype sst2A in cancer tissues: lessons from the comparison of anti-sst2A antibodies with somatostatin receptor autoradiography.

The somatostatin receptor sst2A is highly expressed at the cellular surface of neuroendocrine and other human tumor cells, allowing somatostatin receptor-targeted scintigraphic tumor imaging and tumor therapy. In vitro information on tumoral somatostatin receptor expression is provided mainly by receptor autoradiography, based on binding of radiolabeled somatostatin analogs to tumoral somatostatin receptors. Recent availability of anti-sst2A antibodies opens the possibility of sst2A assessment in human tumors with immunohistochemistry. The aim of the present study was to evaluate the usefulness of several commercial anti-sst2A antibodies for this purpose in comparison with the extensively characterized antibody R2-88 and with receptor autoradiography. sst2A immunohistochemistry was performed in 64 formalin-fixed tumors known to frequently express sst2A (pancreatic, gastrointestinal, pulmonary neuroendocrine tumors, breast carcinomas, meningiomas, pituitary adenomas, neuroblastomas, medulloblastomas, pheochromocytomas, and paragangliomas); receptor autoradiography could be performed simultaneously in 37 of these cases. The commercial antibody SS-800 clearly identified sst2A and correlated excellently with R2-88: compared with R2-88, SS-800 immunohistochemistry generally yielded the same tissular and subcellular staining distribution. Results of R2-88 and SS-800 immunohistochemistry correlated excellently with those obtained with receptor autoradiography; compared with receptor autoradiography, immunohistochemistry with both R2-88 and SS-800 resulted in a slightly lower tumoral sst2A incidence, but a higher resolution, with frequent identification of heterogeneous sst2A distribution at high magnification. Finally, not only membranous, but also cytoplasmic, sst2A immunostaining was simultaneously observed with both antibodies in some tumors. In conclusion, the commercially available SS-800 antibody promises to be useful for the routine immunohistochemical assessment of sst2A in formalin-fixed human tumors.

Amino Acids↗
PubMed1999

[Autoradiography of neurotransmitter receptors in whole human brain hemisphere sections].

Autoradiography is one of our most important tools to gain knowledge about neurotransmitter-receptors playing a key-role in information transmission between neurons. Autoradiography, in its most sophisticated form, is performed on whole human hemispheric sections. The main objective of the authors is to present this application of autoradiography. This in vitro method produces images with high spatial resolution that enable us to qualitatively and quantitatively characterize the regional distribution of the receptors under study. With this technique both the different receptor systems in various physiological and pathological conditions of the brain and the pharmacological parameters of the radioligand, itself, used for a given investigation can be analysed. As a consequence, the results of autoradiography can be successfully used in drug development and trial, brain research and, indirectly, in the every day practice of physicians (diagnosis, differentialdiagnosis, therapy). Autoradiography plays an important role in the validation of in vivo techniques (positron emission tomography, single photon emission tomography) and results in a more complex (in vivo and in vitro) insight into the neurochemical organisation of the brain.

Autoradiography↗