Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes

SummaryRecombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ per-fused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.

Nuclear and Extranuclear Estrogen Binding Sites in the Rat Forebrain and Autonomic Medullary Areas

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating 125I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting 125I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of 125I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting 125I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received 125I-estradiol combined with cold estradiol. The association of 125I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

Tissue Uptake of Circulating Thrombopoietin Is Increased in Immune-Mediated Compared With Irradiated Thrombocytopenic Mice

We have previously demonstrated a significant inverse correlation between circulating thrombopoietin (TPO) levels and peripheral platelet (PLT) counts in patients with thrombocytopenia secondary to megakaryocytic hypoplasia but not in patients with immune thrombocytopenic purpura (ITP; Chang et al, Blood 88:3354, 1996). To test the hypothesis that the differences in the circulating TPO levels in these two types of thrombocytopenia are caused by differences in the total capacity of Mpl receptor-mediated TPO clearance, thrombocytopenia was induced in female CD-1 mice either by sublethal irradiation (irradiated) or rabbit antimouse PLT serum (RAMPS) for 1 day (1 d RAMPS) and 5 days (5 d RAMPS). A well-characterized murine model of autoimmune thrombocytopenic purpura, male (NZW × BXSB) F1 mice (W/B F1), was also included in this study. All thrombocytopenic mice and their controls received trace amounts of 125I-recombinant murine TPO (125I-rmTPO) intravenously and were killed 3 hours postinjection. Blood cell-associated radioactivity was significantly decreased in all 4 groups of thrombocytopenic mice. Significantly increased plasma and decreased whole spleen-associated radioactivity was observed in the irradiated group compared with controls (P < .05). While a lesser but still significant increase in plasma and decrease in whole spleen-associated radioactivity was observed in the 1 d RAMPS mice (P < .05), there were no significant differences between the 5 d RAMPS nor the W/B F1 male mice compared with controls, although whole spleen-associated radioactivity was higher in the W/B F1male. A significant inverse correlation of plasma and whole spleen-associated radioactivity was demonstrated in W/B F1male mice (r = −.91, n = 6, P < .05). There was also a decrease in bone (femur)/blood-associated radioactivity in the irradiated group compared with controls (P < .05), but a significant increase in 1 d and 5 d RAMPS mice (P < .01). Furthermore, the 125I-rmTPO uptake capacity within the spleen and marrow of immune thrombocytopenic mice appeared to be associated with a higher megakaryocytic mass when tissue samples were examined by light microscopy. Internalization of 125I-rmTPO by megakaryocytes and PLTs in the spleens and marrows of ITP mice was also demonstrated directly using electron microscopic autoradiography. Labeled PLTs were also found within splenic macrophages. Additionally, the mean PLT volumes of RAMPS mice were significantly higher than those of the control and irradiated mice (P < .05), as was the bound 125I-rmTPO (cpm) per million PLT (P < .05). Finally, significantly decreased 125I-rmTPO degradation products were only found in the plasma of the irradiated mice compared with control animals (P < .05). These data suggest that the lack of Mpl+ cells in the mice with thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated) results in decreased uptake and degradation of TPO and higher circulating TPO levels. Furthermore, these data also suggest that, after a brief TPO surge in response to immune thrombocytopenia (1 d RAMPS), the lack of an inverse correlation of circulating TPO with PLT counts during steady-state immune thrombocytopenic mice (5 d RAMPS + W/B F1 male) is due, at least in part, to its uptake and degradation by the high PLT turnover and increased mass of megakaryocytes.

Tissue Uptake of Circulating Thrombopoietin Is Increased in Immune-Mediated Compared With Irradiated Thrombocytopenic Mice

We have previously demonstrated a significant inverse correlation between circulating thrombopoietin (TPO) levels and peripheral platelet (PLT) counts in patients with thrombocytopenia secondary to megakaryocytic hypoplasia but not in patients with immune thrombocytopenic purpura (ITP; Chang et al, Blood 88:3354, 1996). To test the hypothesis that the differences in the circulating TPO levels in these two types of thrombocytopenia are caused by differences in the total capacity of Mpl receptor-mediated TPO clearance, thrombocytopenia was induced in female CD-1 mice either by sublethal irradiation (irradiated) or rabbit antimouse PLT serum (RAMPS) for 1 day (1 d RAMPS) and 5 days (5 d RAMPS). A well-characterized murine model of autoimmune thrombocytopenic purpura, male (NZW × BXSB) F1 mice (W/B F1), was also included in this study. All thrombocytopenic mice and their controls received trace amounts of 125I-recombinant murine TPO (125I-rmTPO) intravenously and were killed 3 hours postinjection. Blood cell-associated radioactivity was significantly decreased in all 4 groups of thrombocytopenic mice. Significantly increased plasma and decreased whole spleen-associated radioactivity was observed in the irradiated group compared with controls (P < .05). While a lesser but still significant increase in plasma and decrease in whole spleen-associated radioactivity was observed in the 1 d RAMPS mice (P < .05), there were no significant differences between the 5 d RAMPS nor the W/B F1 male mice compared with controls, although whole spleen-associated radioactivity was higher in the W/B F1male. A significant inverse correlation of plasma and whole spleen-associated radioactivity was demonstrated in W/B F1male mice (r = −.91, n = 6, P < .05). There was also a decrease in bone (femur)/blood-associated radioactivity in the irradiated group compared with controls (P < .05), but a significant increase in 1 d and 5 d RAMPS mice (P < .01). Furthermore, the 125I-rmTPO uptake capacity within the spleen and marrow of immune thrombocytopenic mice appeared to be associated with a higher megakaryocytic mass when tissue samples were examined by light microscopy. Internalization of 125I-rmTPO by megakaryocytes and PLTs in the spleens and marrows of ITP mice was also demonstrated directly using electron microscopic autoradiography. Labeled PLTs were also found within splenic macrophages. Additionally, the mean PLT volumes of RAMPS mice were significantly higher than those of the control and irradiated mice (P < .05), as was the bound 125I-rmTPO (cpm) per million PLT (P < .05). Finally, significantly decreased 125I-rmTPO degradation products were only found in the plasma of the irradiated mice compared with control animals (P < .05). These data suggest that the lack of Mpl+ cells in the mice with thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated) results in decreased uptake and degradation of TPO and higher circulating TPO levels. Furthermore, these data also suggest that, after a brief TPO surge in response to immune thrombocytopenia (1 d RAMPS), the lack of an inverse correlation of circulating TPO with PLT counts during steady-state immune thrombocytopenic mice (5 d RAMPS + W/B F1 male) is due, at least in part, to its uptake and degradation by the high PLT turnover and increased mass of megakaryocytes.

Mechanism of Peptide-induced Mast Cell Degranulation

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription–-PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100–500 s at concentrations of substance P from 50 to 5 μM). Degranulation in response to intracellularly applied substance P was inhibited by GDPβS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein α subunits subsequent to their translocation across the plasma membrane.

Intracellular electrolyte levels and transport of secretory granules in exocrine gland cells

We demonstrate the intracellular transport of secretory granules of a silk protein, fibroin, from the Golgi region to the apical cytoplasm with special reference to microtubule organization, electrolyte concentrations and the acidic intragranular pH of normal and mutant posterior silk gland cells, using the techniques of electrophysiological microelectrode and microprobe analysis and of light and electron microscopic autoradiography. The silk gland cells of a recessive mutant making only flimsy cocoons were defective in the microtubule systems, did not stain with an anti-tubulin antibody in immunofluorescent microscopy, and accumulated intracellular granules in the apical and basal cytoplasm. The increase in intracellular calcium concentration and levels of chloride secretion were also reduced in the mutant cells. A carboxylic ionophore, monensin, which collapsed the granular H+ gradient, induced the transport of chloride and an increase in the intracellular calcium concentration, while it blocked the intracellular transport of granules from the Golgi region to the apical cytoplasm in normal cells. Thus, we conclude that the H+ gradient across the membrane of secretory granules is responsible for the intracellular transport of the secretory granules along the microtubule systems in silk gland cells, while Ca2+ is thought to be required for the exocytosis of the granules.