Radioimagers as an alternative to film autoradiography forin situ quantitative analysis of125I-ligand receptor binding and pharmacological studies

Summary Three radioimagers, the μ-imager, the β-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacologicalin situ quantitative analysis. Two iodinated ligands¹²⁵I-interleukin-1α and¹²⁵I-gonadotropin releasing hormone agonist, were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical γ detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the μ-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and β-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion forin situ quantitative studies on tissue sections. They combine precise imaging forin situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, broughtin situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.     

MICROSCOPIC DETERMINATION OF BONE PHOSPHORUS BY QUANTITATIVE AUTORADIOGRAPHY OF NEUTRON-ACTIVATED SECTIONS

Longitudinal sections of human cortical bone were submitted to thermal neutrons. γ-ray spectra were recorded repeatedly during 15 days following irradiation. They showed that Na²⁴ is predominant as early as 3 hours after activation and that all the γ-emitters have decayed on the 15th day. When the γ-rays have disappeared, β-rays are still produced by the sections. It was proved by the absorption curve in aluminium that all these β-rays are issued from the P³² induced in the sections by activation of P³¹. Therefore autoradiograms registered 15 days after activation reveal the distribution of P³² in the sections. γ-ray spectra and β-ray absorption curves of neutron activated sections of ivory demonstrated a mineral composition similar to that of bone. Autoradiograms of ivory sections activated for various times were used to establish the relation between the optical density of the autoradiograms and the radioactivity in P³². When the bone autoradiograms are compared with the ivory standards of known radioactivity, the optical densities of single osteons (Haversian systems), can be related to their phosphorus contents. Autoradiograms and microradiograms of the same sections were examined side by side. The least calcified osteons, that contain 80 per cent of the calcium of the fully calcified osteons, also contain about 80 per cent of the phosphorus of the fully mineralized osteons. It is concluded that the Ca:P ratio remains constant while mineralization of bone tissue is being completed.     

A quantitative ''in-situ'' hybridization method using computer-assisted image analysis. Validation and measurement of atrial-natriuretic-factor mRNA in the rat heart.

We have validated a quantitative 'in-situ' hybridization method and computer-assisted image analysis of autoradiographs as a technique for measuring atrial-natriuretic-factor (ANF) mRNA in tissue sections of rat heart by: (i) producing radioactive standards to calibrate the autoradiograms and (ii) assessing: (a) specificity (through RNAase A background subtraction, comparison of ANF mRNA and non-ANF mRNA probe binding to sections, Northern analysis and section-thickness titration curves); (b) sensitivity (by calculating the limit of detection for ventricular levels of ANF mRNA); (c) precision [inter-assay CV (coefficient of variation) less than 10%; intra-assay CV 6-7%]; and (d) accuracy. We have found with this technique that deoxycortone and saline treatment of rats elevates ANF mRNA to a larger extent in the ventricles than in the atria and that, in neonatal-rat hearts, ANF mRNA is elevated in all cardiac chambers relative to adult levels.     

Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues

1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.     

Auto-radiographic determination of S35 in tissues after injection of methionine-S35 and sodium sulfate-S35

The loss of "bound" S(35) that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S(35) or Na(2)S(35)O(4). Valid autoradiography of bound S(35) in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S(35) is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S(35) given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S(35).     

Preparation of iodinated standards for quantitative radioautography in vitro using a tritium-sensitive film

In the present study we developed iodinated 125I-standards and tested the response of tritium-sensitive sheet film (3H-Ultrofilm) to varying concentrations of 125I prepared from brain "paste" mixed with mono-iodinated TiTx gamma toxin. Results have practical implications since they allow quantitative measurements of autoradiograms obtained with 125I-coordinats. An example is shown for 125I-neurotensin binding sites on rat brain sections.     

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.     

The immunocytochemical demonstration of Toxoplasma antigen in the brains of congenitally infected mice

The peroxidase anti-peroxidase immunocytochemical staining method was used to identify Toxoplasma antigen in paraffin embedded sections of the brains of 22 mice congenitally infected with the parasite. Intact Toxoplasma tissue cysts were readily demonstrated in the brain in all cases. In 4 of the 22 infected mice there was evidence of rupture of the cyst wall and/or presence of extra-cystic Toxoplasma antigen. Further support for the extra-cystic location of Toxoplasma antigen was obtained by electron microscopy of reprocessed tissue which revealed endozoites in the area immediately surrounding a ruptured cyst. The possible implications of these findings in relation to the pathogenesis of congenital toxoplasmic meningo-encephalitis are discussed.     

A new antigen retrieval technique for human brain tissue

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.     

AUTORADIOGRAPHIC DETERMINATION OF S35 IN TISSUES AFTER INJECTION OF METHIONINE-S35 AND SODIUM SULFATE-S35

The loss of "bound" S³⁵ that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S³⁵ or Na₂S³⁵O₄. Valid autoradiography of bound S³⁵ in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S³⁵ is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S³⁵ given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S³⁵.     

Quantitative plutonium microdistribution in bone tissue of vertebra from occupationally exposed worker

The purpose of this work is the receiving of quantitative data on Pu microdistribution in different structural elements of human bone tissue for local dose assessment and dosimetric models validation. Thoracic vertebra sample was taken for the study from former Mayak worker with rather high Pu burden, including information on occupational and exposure history, medical information and data on Pu content in organs. Lexan film autodiagrams were obtained using method of neutron-induced autoradiography from bone tissue sections. Quantitative analysis of randomly selected vision fields on one of autoradiograms was performed: fission fragment tracks Pu in different bone tissue areas were calculated, surface of bone tissue areas were defined. Quantitative information on Pu microdistribution in human bone tissue was obtained for the first time. On the basis of obtained data quantitative relation of Pu decays in bone volume to decays on bone surface in cortical and trabecular fractions were defined as 2.0 and 0.4, correspondingly. Actual quantitative relation of decays in bone volume to decays on bone surface is significantly different from recommended by ICRP for cortical fraction. Biokinetic model parameters of extrapulmonary ICRP compartment might need to be adjusted after expansion of data set on quantitative Pu microdistribution in other bone types in human that will involve new cases with different exposure pattern of radionuclide.     

Variability of laminin immunoreactivity in human autopsy brain

Summary Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Autoradiographic evaluation of [11C]vinpocetine binding in the human postmortem brain

The main objective ofthe study was to evaluate with autoradiographic technique whether or not [11C]vinpocetine, a compound widely used in the prevention and treatment of cerebrovascular diseases (Cavinton, Gedeon Richter Ltd., Budapest), binds to specific sites in the human brain in post mortem human brain sections. Binding was assessed under four conditions: the incubation was performed using Tris-HCl buffer with or without the addition of salts (0.1% (weight/vol) ascorbic acid, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2), with or without the addition of excess (10 microM) unlabelled vinpocetine. Measurements on digitized autoradiograms indicated that [11C]vinpocetine labelled all grey matter areas in the human brain to a similar extent and no significantly heterogeneous binding could be demonstrated among cortical or subcortical regions. The addition of excess unlabelled vinpocetine lowered the binding slightly in all regions. Although these results indicate that [11C]vinpocetine does not bind to human brain transmitter receptors or transporters with a high affinity (Ki < 10 nM), it cannot be ruled out that the compound binds to receptors and/or transporters with lower affinity.     

Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections

MALDI MS imaging has been extensively used to produce qualitative distribution maps of proteins, peptides, lipids, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. There is growing demand to quantify the amount of target compounds in the tissue sections of different organs. We present a novel MS imaging software including protocol for the quantitation of drugs, and for the first time, an endogenous neuropeptide directly in tissue sections. After selecting regions of interest on the tissue section, data is read and processed by the software using several available methods for baseline corrections, subtractions, denoising, smoothing, recalibration and normalization. The concentrations of in vivo administered drugs or endogenous compounds are then determined semi-automatically using either external standard curves, or by using labeled compounds, i.e., isotope labeled analogs as standards. As model systems, we have quantified the distribution of imipramine and tiotropium in the brain and lung of dosed rats. Substance P was quantified in different mouse brain structures, which correlated well with previously reported peptide levels. Our approach facilitates quantitative data processing and labeled standards provide better reproducibility and may be considered as an efficient tool to quantify drugs and endogenous compounds in tissue regions of interest.     

Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin

The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them.     

Autoradiographic Differentiation of Free, Bound, Pure, and Impure Thymidine-3H

Intestinal tissue from animals injected with tritium-labeled thymidine were examined by two autoradiographic methods. Conventional autoradiograms using fixed, solvent-dehydrated, paraffin-embedded tissues, and wet mounted, were compared with an autoradiographic method designed for localizing diffusible substances utilizing dry-mounted, freeze-dried frozen sections. The autoradiograms prepared by conventional methods show more than 95% of the activity located over the nucleus while, autoradiograms of intestinal tissue from the same animal prepared by the freeze-dried method shows only 88% of the activity over the nucleus. Animals injected with impure thymidine-³H, containing self-radiolysis decomposition products due to storage, led to significant alteration in the autoradiographic pattern, particularly those prepared by the freeze-dried method which show only 52% of the activity over the nucleus. Thus, conventional treatment of tissue extracted free unincorporated thymidine, metabolic products of thymidine-³H, and impurities of thymidine-³H. However, autoradiograms prepared by the dry-mounted freeze-dried method retained all the label.     

Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase

Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.     

Radioautographic and quantitative study of insulin binding sites in the rat brain

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.     

Selective adherence of lymphocytes to myelinated areas of rat brain.

An in vitro system developed for studying lymphocyte binding to high endothelial venules (HEV) of lymph nodes was used to determine if there are similar binding sites in other organs of the rat. Thoracic duct lymphocytes (TDL) adhered selectively and uniformly to white matter when overlaid onto glutaraldehyde-fixed tissue sections of cerebellum and cerebrum. The pattern of TDL adherence to cerebellar sections showed that binding to nonmyelinated areas was negligible. Comparison of TDL-white matter to TDL-HEV binding demonstrated that the density of adherence to each site was quantitatively similar. In contrast, lymphocytes exhibited little tendency to bind to tissue sections of liver, spleen, heart, thymus, and salivary glands. TDL adherence to cerebellar white matter occurred rapidly, was cell dose dependent and optimal at 7 degrees C. White matter binding was also a property of spleen lymphocytes but the thymus was deficient in cells with this capability. The affinity of TDL and spleen lymphocytes for myelinated areas of the brain suggests the presence of myelin binding receptors on these cells.