In vivo labeling of resident peritoneal macrophages

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident M phi were labeled by both the green dye and the red Mab markers, while recruited M phi or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited M phi in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or M phi mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the M phi, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.     

In vivo labeling with1251-CRH of human ACTH-producing pituitary adenomas heterotransplanted to nude mice

Tissue from 23 pituitary adenomas causing Cushing’s disease was implanted subcutaneously into 159 NuNu/NMRi mice, resected after 21 or 35 days, and evaluated histologically and immunohistochemically. After 21 days, 74.3% of the grafts survived, 59% having less than 30% necrotic adenoma cells. After 35 days, 45% of the adenoma fragments survived, 37% having less than 30% necrotic adenoma cells. The preservation of the grafts was essentially dependent on the grade of vascularization accomplished by migration of the host’s capillaries. As assessed by adrenal weight and histologically, biological activity of the transplants could not be detected. Histologically, the grafts maintained the features of their primary tumors, and adrenocorticotropic hormone (ACTH) could be visualized immunohistologically.Seventeen mice with subsequently proved preserved adenoma tissue received an intravenous injection of 12.5 μCi¹²⁵l-corticotropin-releasing hormone (CRH) and light microscopy-autoradiography was performed. Specific labeling, as verified by positive and negative controls, was exhibited by 1 1 of 15 transplants originating from 3 highly differentiated ACTH cell adenomas. Four did not label clearly positive. Two grafts of an undifferentiated mucoid cell pituitary adenoma did not show any labeling.The nude mouse model is a useful tool for the study of ACTH-producing pituitary adenomas in vivo. Highly differentiated ACTH cell adenomas can be labeled with radioactive CRH in vivo.     

In vivo quantum dot labeling of mammalian stem and progenitor cells.

Fluorescent semiconductor nanocrystal quantum dots (QDs) are a class of multifunctional inorganic fluorophores that hold great promise for clinical applications and biomedical research. Because no methods currently exist for directed QD-labeling of mammalian cells in the nervous system in vivo, we developed novel in utero electroporation and ultrasound-guided in vivo delivery techniques to efficiently and directly label neural stem and progenitor cells (NSPCs) of the developing mammalian central nervous system with QDs. Our initial safety and proof of concept studies of one and two-cell QD-labeled mouse embryos reveal that QDs are compatible with early mammalian embryonic development. Our in vivo experiments further show that in utero labeled NSPCs continue to develop in an apparent normal manner. These studies reveal that QDs can be effectively used to label mammalian NSPCs in vivo and will be useful for studies of in vivo fate mapping, cellular migration, and NSPC differentiation during mammalian development.     

In vitro and in vivo oxidative stress associated with Alzheimer's amyloid beta-peptide (1-42)

The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.     

In vivo receptor labeling of peripheral benzodiazepine receptor by ex vivo binding of [3H]PK11195

In vivo receptor labeling of the peripheral benzodiazepine receptor was investigated using ex vivo binding of [3H]-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carb ox-amide ([3H]PK11195). In autoradiographic studies, high level specific binding of [3H]PK11195 was observed in the olfactory bulb. Intravenous administration of PK11195 dose-dependently (0.03-3 mg/kg) inhibited ex vivo binding of [3H]PK11195 in the olfactory bulb. Likewise, N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (DAA1106), a newly identified peripheral benzodiazepine receptor-specific ligand, dose-dependently (0.1-100 mg/kg) reduced ex vivo binding of [3H]PK11195, when administered intraperitoneally. In contrast, clonazepam, a central benzodiazepine receptor-specific agonist, had negligible effects on ex vivo binding of [3H]PK11195. We propose that the ex vivo receptor binding technique we used will facilitate determination of in vivo receptor occupancy of the peripheral benzodiazepine receptor.     

In vivo biocompatibility studies. V. In vivo leukocyte interactions with Biomer

A cage implant system was utilized to quantitatively and qualitatively characterize in vivo leukocyte interactions with cast Biomer. Scanning electron microscopy (SEM) in conjunction with cytochemical staining procedures were used to investigate the cellular events at the leukocyte/Biomer interface as well as in the inflammatory exudate over a 21-day implantation period. SEM was used to characterize leukocyte morphology on the Biomer surface and the cytochemical stains were used to differentially count leukocytes and to demonstrate intracellular alkaline and acid phosphatase activity. The results showed that the population density of leukocytes on the Biomer surface diminished with implantation time. The population density of multinucleated foreign body giant cells remained constant with time, while the numbers of nuclei per giant cell increased. The differential analysis revealed that macrophages preferentially adhered to the Biomer surface compared to other leukocytes in the exudate. The phagocytic capability of all adherent leukocytes, including giant cells, decreased with time and this corresponded to changes in leukocyte morphology observed with SEM.     

Biosynthesis of collagen crosslinks. III. In vivo labeling and stability of lung collagen in rats with bleomycin-induced pulmonary fibrosis

Rats were injected intraperitoneally with 1 mCi (each) of [3H]lysine at Day 11 of neonatal life to label their lung collagen. Five weeks later, half of the animals were given an intratracheal injection of 1.5 U of bleomycin sulfate via a tracheostomy; control animals received saline intratracheally by the same technique. Age-matched groups of control and bleomycin-treated rats were killed, and their lung collagen was analyzed at zero (control animals only), 1, 2, 4, 6, and 10 wk after bleomycin administration, a time course appropriate for development of pulmonary fibrosis in this animal model. We measured radioactivity in hydroxylysine and in the difunctional collagen crosslinks hydroxylysinonorleucine and dihydroxylysinonorleucine at each time point. No evidence of breakdown of this pool of mature, preformed collagen was observed in lungs of either the control or the bleomycin-treated rats. We also measured the total lung content of hydroxypyridinium, a trifunctional collagen crosslink, by its intrinsic fluorescence. There was no evidence of collagen degradation in lungs of either group of rats by this criterion either. We conclude that there is no biochemically detectable turnover of mature lung collagen, defined as that pool of lung collagen that is obligatorily extracellular (i.e., crosslinked and containing labeled hydroxylysine from an injection of precursor 5 to 15 wk earlier), in either normal rat lungs or lungs of rats made fibrotic with bleomycin. Statistical analysis of the data suggests that our methodology was sensitive and precise enough to have detected turnover of less than 0.5% of lung collagen per day, some 20-fold less than estimates of lung collagen turnover that have been suggested to be occurring in vivo by others using different techniques and presumably studying different pools of lung collagen.     

In vivo experiments with a pH-ISFET electrode

The_{in vivo} stability and pH sensitivity of 19 intravascular pH electrodes for continuous monitoring have been investigated. The sensors were mounted in indwelling vascular catheters (7F) which were inserted into the arteries of seven anaesthetised and mechanically ventilated dogs. Variations in arterial pH, ranging from 6·82 to 7·72, were obtained by infusion of sodium bicarbonate and hydrochloric acid and by hyper/ hypoventilation with various volume fractions of carbon dioxide in the inspiratory gas mixture. The pH sensor output potential was compared within vitro pH determinations of arterial blood samples. After an initial stabilisation period following their introduction into the arterial blood, the electrodes showed an average long-term drift of 2·3 mVh⁻¹. When this drift was taken into account, a typical pH sensitivity of 50 mV per pH was found. The relationship between the electrode potentials and thein vitro pH values was linear and in almost all cases the correlation coefficient (r) was above 0·9. The electrodes responded rapidly enough to reflect breath-to-breath oscillations in pH.     

Immunogold labeling of cerebrovascular and neuritic plaque amyloid fibrils in Alzheimer's disease with an anti-beta protein monoclonal antibody

A monoclonal antibody raised to a synthetic peptide consisting of residues 8 to 17 of the amyloid beta protein of Alzheimer's disease was employed for immunogold electron microscopic studies on amyloid fibrils of cerebrovascular walls and neuritic plaques in this disease. Electron microscopy revealed a specific gold labeling of the amyloid fibrils in these structures. This provides ultrastructural evidence that beta protein is intimately associated with the amyloid fibril. With previous chemical evidence, this observation supports the concentration that it is an intrinsic component of the fibril.     

Beta amyloid angiogenic activity in vitro and in vivo

Angiogenesis has been suggested as a direct contributor to Alzheimer's disease (AD) pathology. The major pathological hallmarks of AD are the presence of neurofibrillary tangles and, beta-amyloid plaques associated with activated microglia, astrocytes, degenerating neurons and vascular toxicity. In this study, Abeta1-40 and Abeta1-42 peptides, both components of the senile plaques in AD, were used to study their angiogenic activity in vitro, by using normal human cerebral endothelial cells (HCECs), and in vivo, by using the chick embryo chorioallantoic membrane (CAM) assay. Results showed that both peptides stimulate in vitro endothelial cell proliferation, chemotaxis and morphogenesis in Matrigel. Moreover, by using the aorta ring assay, both peptides stimulated the formation of capillary-like structures. An angiogenic response was induced in the CAM assay, similar to that induced by fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. Overall, these data support the hypothesis that Abeta peptides may contribute to angiogenesis occurring in AD and suggest that limiting the pro-angiogenic activity of Abeta peptides may therefore provide a useful target to control angiogenesis associated to AD and therefore limit the disease progression.     

In vivo experiments with tracheostoma tissue connector prototypes

In cancer patients who have undergone total surgical removal of the larynx, ideally voice rehabilitation should be performed using a shunt valve (placed in a fistula of the tracheo-esophageal wall) and a tracheostoma valve (TSV) to enable hands-free tracheo-esophageal speech. A tracheostoma is created by suturing the trachea into the lower anterior part of the neck, and a TSV is a device that can be placed at the stoma. Unfortunately, many patients are unable to use a TSV, mainly due to fixation difficulties. To improve the fixation of the TSV, tracheostoma tissue connector (TS-TC) prototypes have been designed. Prototype 1 consisted of a titanium ring, inner diameter 30 mm, with a circular polypropylene mesh glued to it with silicone adhesive. Four holes had been drilled into the ring for the insertion of sub- and percutaneous screws. Prototype 2 consisted of a silicone rubber ring, inner diameter 30 mm, combined with polypropylene mesh and four titanium inserts that functioned as a base plate for the insertion of sub- and percutaneous screws. In adult female goats a tracheostoma was created and the prototypes were implanted. After 6 weeks of subcutaneous implantation, percutaneous screws were inserted. After twelve weeks, the experiment was terminated and the implants with the surrounding tissues were processed and examined histologically. The clinical appearance during weeks 7-12 varied from very poor to relatively good. Histologically, the implants showed a uniform inflammatory response. We found that all the tissue surrounding the screws showed signs of epithelial down growth. It was concluded that the two-stage implantation procedure of our prototype TS-TCs in this animal model was unsuccessful. Additional research efforts are necessary to improve tissue immobilization and to devise reliable fixation systems for TSVs.     

In vivo cell interactions with calcium phosphate bioceramics

Biointegration, resorption process, and solubility in physiological environments of calcium phosphate materials are scarcely described by ultrastructural studies. In vivo cells interactions with calcium phosphate materials are scarcely described by ultrastructural studies. In vivo cells interactions with calcium phosphate biomaterials are mediated by different proteins from physiological fluid, and in order to observe at the ultrastructural level the cell colonization, the resorption, process and the biointegration, we used in these experiments calcium phosphate materials precoated with fibronectin or not precoated. Two kinds of well determined materials were used for this study, Beta-tricalcium phosphate (B-TCP) and hydroxyapatite (HAP). The implants were soaked in human fibronectin diluted solution and were implanted in the connective tissue of rabbit abdomen. Our results showed that the fibroblasts and macrophagous++ cells interaction with the calcium phosphate crystal (B-TCP and HAP) was more important in the experiments with a fibronectin bilayer. In the presence of fibronectin at the grains surface of the material, cystic cavities' or fibrous encapsulation was suppressed and cells with fibers were in close contact with the material. The presence of fibronectin immediately after implantation seemed to increase the adhesion and the cell colonization. Fibronectin creates an organic interface between crystals and cells, and can promote interactions from cells and biomaterials.     

In vivo genotoxicity studies with p-benzoquinone dioxime.

P-Benzoquinone dioxime (BQD) appears to be a sex-specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct-acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test. BQD did, however, induce a marked effect upon S-phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell-proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen.     

在不同组织中淀粉样物质5种染色法的比较

目的：探寻淀粉样物质更好的特殊染色方法。方法：采用甲醇刚果红、甲基紫、Bennhola氏刚果红、Freudenthal氏刚果红、碱性刚果红5种染色,显微镜下观察组织切片中淀粉样蛋白的染色情况。结果：喉部组织、皮肤组织、肾穿刺组织经甲醇刚果红、甲基紫、Bennhola氏刚果红、Freudenthal氏刚果红、碱性刚果红染色方法分别将淀粉样物质均染成红色但其鲜艳程度不同,背景着色程度不同。结论：喉部组织、皮肤组织、肾穿刺组织在淀粉样物质选择特殊染色方法时甲醇刚果红是阳性着色部位最准确、色彩最鲜明、背景最干净的方法。