Morphological Effects and In Vitro Biological Mechanisms of Radiation-Induced Cell Killing by Gold Nanomaterials.

Gold nanomaterials have been shown to augment radiation therapy both in vitro and in vivo. However, studies on these materials are mostly phenomenological due to nanoparticle heterogeneity and the complexity of biological systems. Even accurate quantification of the particle dose still results in bulk average biases; the effect on individual cells is not measured but rather the effect on the overall population. To perform quantitative nanobiology, we coated glass coverslips uniformly at varying densities with Au nanoparticle preparations with different morphologies (45 nm cages, 25 nm spheres, and 30 nm rods). Consequently, the effect of a specific number of particles per unit area in contact with breast cancer cells growing on the coated surfaces was ascertained. Gold nanocages showed the highest degree of radiosensitization on a per particle basis, followed by gold nanospheres and gold nanorods, respectively. All three materials showed little cytotoxic effect at 0 Gy, but clonogenic survival decreased proportionally with the radiation dose and particle coverage density. A similar trend was seen in vivo in the combined treatment antitumor response in 4T1 tumor-bearing animals. The presence of gold affected the type and quantity of reactive oxygen species generated, specifically superoxide and hydroxyl radicals, and the concentration of nanocages correlated with the development of more numerous double-stranded DNA breaks and increased protein oxidation as measured by carbonylation. This work demonstrates the dependence on morphology and concentration of radiation enhancement by gold nanomaterials and may lead to a novel method to differentiate intra- and extracellular functionalities of gold nanomedicine treatment strategies. It further provides insights that can guide the rational development of gold nanomaterial-based radiosensitizers for clinical use.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Parthenolide induces rapid thiol oxidation that leads to ferroptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is both a devastating and common disease. Every year in the United States, about 24,500 men and 10,000 women are diagnosed with HCC, and more than half of those diagnosed patients die from this disease. Thus far, conventional therapeutics have not been successful for patients with HCC due to various underlying comorbidities. Poor survival rate and high incidence of recurrence after therapy indicate that the differences between the redox environments of normal surrounding liver and HCC are valuable targets to improve treatment efficacy. Parthenolide (PTL) is a naturally found therapeutic with anti-cancer and anti-inflammatory properties. PTL can alter HCC's antioxidant environment through thiol modifications leaving tumor cells sensitive to elevated reactive oxygen species (ROS). Investigating the link between altered thiol mechanism and increased sensitivity to iron-mediated lipid peroxidation will allow for improved treatment of HCC. HepG2 (human) and McARH7777 (rat) HCC cells treated with PTL with increasing concentrations decrease cell viability and clonogenic efficiency in vitro. PTL increases glutathione (GSH) oxidation rescued by the addition of a GSH precursor, N-acetylcysteine (NAC). In addition, this elevation in thiol oxidation results in an overall increase in mitochondrial dysfunction. To elucidate if cell death is through lipid peroxidation, using a lipid peroxidation sensor indicated PTL increases lipid oxidation levels after 6 h. Additionally, western blotting reveals glutathione peroxidase 4 (GPx4) protein levels decrease after treatment with PTL suggesting cells are incapable of preventing lipid peroxidation after exposure to PTL. An elevation in lipid peroxidation will lead to a form of cell death known as ferroptosis. To further establish ferroptosis as a critical mechanism of death for HCC in vitro, the addition of ferrostatin-1 combined with PTL demonstrates a partial recovery in a colony survival assay. This study reveals that PTL can induce tumor cell death through elevations in intracellular oxidation, leaving cells sensitive to ferroptosis.

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Photothermal Response Induced by Nanocage-Coated Artificial Extracellular Matrix Promotes Neural Stem Cell Differentiation.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

Actinomycin-D and dimethylamino-parthenolide synergism in treating human pancreatic cancer cells.

Preclinical Research & Development Pancreatic cancer is the third leading cause of death in the US with a poor 5-year survival rate of 8.5%. A novel anti-cancer drug, dimethylamino parthenolide (DMAPT), is the water-soluble analog of the natural sesquiterpene lactone, parthenolide. The putative modes of action of DMAPT are inhibition of the Nuclear chain factor kappa-light-chain enhancer of activated B cells (NFκB) pathway and depletion of glutathione levels; the latter causing cancer cells to be more susceptible to oxidative stress-induced cell death. Actinomycin-D (ActD) is a polypeptide antibiotic that binds to DNA, and inhibits RNA and protein synthesis by inhibiting RNA polymerase II. A phase 2 clinical trial indicated that ActD could be a potent drug against pancreatic cancer; however, it was not a favored drug due to toxicity issues. New drug entities and methods of drug delivery, used alone or in combination, are needed to treat pancreatic cancer more effectively. Thus, it was postulated that combining DMAPT and ActD would result in synergistic inhibition of Panc-1 pancreatic cancer cell growth because DMAPT's inhibition of NFκB would enhance induction of apoptosis by ActD, via phosphorylation of c-Jun, by minimizing NFκB inhibition of c-Jun phosphorylation. Combining these two drugs induced a higher level of cell death than each drug alone. A fixed drug ratio of DMAPT: ActD (1,200:1) was used. Data from metabolic (MTT) and colony formation assays were analyzed for synergism with CompuSyn software, which utilizes the Chou-Talalay equation. The analyses indicated synergism and moderate synergism at combination concentrations of DMAPT/ActD of 12/0.01 and 18/0.015 µM, respectively.

Quantitative microinjection using fluorescence calibration of streaming microdroplets on a superhydrophobic surface.

A simple and reproducible procedure was developed to measure the volume of liquid microinjected into cells. A calibration curve of droplet fluorescence intensity versus volume was constructed by injecting a fluorescent dextran solution through a 125-150 µm diameter micropipette into an oil-filled culture dish to create a spray of varied-sized droplets. The droplets retained a spherical shape because they were in an oil medium and they settled onto a glass surface coated with a superhydrophobic surface. Fluorescent micrographs of the droplets were obtained and analyzed with Image-J software to quantify the fluorescence intensity and radius of each spherical droplet to produce the calibration curve. Subsequently, Dut-145 human prostate carcinoma cells were microinjected with the same fluorescent dextran solution and fluorescent micrographs of the cells were obtained using the identical exposure conditions used to photograph the droplets. The measured fluorescence intensity of the microinjected cells was entered into the formula for the regression line that was fit to the calibration curve allowing determination of the volume of solution injected into each cell. Thus, a mixture consisting of known concentrations of a test material of test material (macromolecules, drugs, etc.) and a fluorescent dextran, volumetric, tracer can be used to quantify the relationship between the amount of a microinjected material and subsequent effects on cells.

The Pathophysiological Sequence of Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Male Mice.

In search of the sequence of pathogenic events leading to glucocorticoid-induced osteonecrosis, we determined the molecular, biomechanical, cellular, and vascular changes in the femur of C57BL/6 mice receiving prednisolone for 14, 28, or 42 days. The femoral head, but not the distal femur, of mice treated for 14 days showed a decrease in the expression of the hypoxia-inducible factor (Hif)-1α and vascular endothelial growth factor (VEGF), the number of osteoblasts, and bone formation rate and strength and showed an increase in osteoclasts. These changes were accompanied by conversion of the normal dendritic vasculature to pools of edema as detected by magnetic resonance imaging, providing robust diagnostic evidence of early osteonecrosis. At that time point, there were no detectable changes in bone density, cortical or cancellous bone architecture, midshaft or distal cancellous bone, or osteocyte apoptosis. In mice treated for 28 days, femoral head cancellous density, cortical width, and trabecular thickness decreased, and by 42 days the femoral heads had full-depth cortical penetrations and cancellous tissue osteonecrosis. These results indicate that the femoral head is a particularly sensitive anatomical site to the adverse effects of glucocorticoid excess on bone and that decreases of Hif-1α and VEGF expression, bone vascularity, and strength precede the loss of bone mass and microarchitectural deterioration, thus rendering the femoral head vulnerable to collapse.

Indole carboxylic acid esters of melampomagnolide B are potent anticancer agents against both hematological and solid tumor cells.

A series of novel, heteroaryl carboxylic acid conjugates of the sesquiterpene melampomagnolide-B (MMB, 3) has been evaluated as antitumor agents against an NCI panel of 64 human hematopoetic and solid tumor cell lines. The indole-3-acrylic acid conjugate 7j and the indole-3-carboxylic acid conjugate 7k were found to be the most potent analogs in the series. Compounds 7j and 7k exhibited remarkable growth inhibition, with GI50 values in the range 0.03-0.30 µM and 0.04-0.28 µM, respectively, against the cell lines in the leukemia sub-panel, and GI50 values of 0.05-0.40 µM and 0.04-0.61 µM, respectively, against 90% of the solid tumor cell lines in the NCI panel. Compound 7a was particularly effective against the sub-panel of breast cancer cell lines with GI50 values in the range <0.01-0.30 µM. Compounds 7j, 7a and its water soluble analog 7p also exhibited potent anticancer activity against rat 9L-SF gliosarcoma cells in culture. Compound 7j was the most potent compound in the series in the M9-ENL1 AML cell assay with a lethal dose concentration EC50 value of 720 nM, and exhibited the greatest cytotoxicity against a collection of primary AML stem cell specimens, which included a specimen that was unresponsive to PTL, affording EC50 values in the range 0.33-1.0 µM in three out of four specimens. The results from this study provide further evidence that analogs of the sesquiterpene MMB can be designed to afford molecules with significantly improved anticancer activity. Thus, both 7j and 7k are considered potential lead molecules in the search for new anticancer agents that can be used as treatments for both hematopoetic and solid tumors.

N-[11CH3]Dimethylaminoparthenolide (DMAPT) uptake into orthotopic 9LSF glioblastoma tumors in the rat.

The aim of this study was to determine the uptake of intravenously administered N-[11CH3]-dimethylaminoparthenolide (DMAPT) into orthotopic 9LSF glioblastoma brain tumors in Fisher 344 rats from positron emission tomography (PET) imaging studies. [11C]methyl iodide (11CH3I) was utilized as a [11C]-labeling reagent to label the precursor methylaminoparthenolide (MAPT) intermediate. From PET imaging studies it was found that brain uptake of N-[11CH3]DMAPT into brain tumor tissue was rapid (30min), and considerably higher than that in the normal brain tissue.

Dimers of Melampomagnolide B Exhibit Potent Anticancer Activity against Hematological and Solid Tumor Cells.

Novel carbamate (7a-7h) and carbonate (7i, 7j, and 8) dimers of melampomagnolide B have been synthesized by reaction of the melampomagnolide-B-triazole carbamate synthon 6 with various terminal diamino- and dihydroxyalkanes. Dimeric carbamate products 7b, 7c, and 7f exhibited potent growth inhibition (GI50 = 0.16-0.99 µM) against the majority of cell lines in the NCI panel of 60 human hematological and solid tumor cell lines. Compound 7f and 8 exhibited anticancer activity that was 300-fold and 1 × 10(6)-fold more cytotoxic than DMAPT, respectively, at a concentration of 10 µM against rat 9L-SF gliosarcoma cells. Compounds 7a-7j and 8 were also screened against M9-ENL1 and acute myelogenous leukemia (AML) primary cell lines and exhibited 2- to 10-fold more potent antileukemic activity against M9-ENL1 cells (EC50 = 0.57-2.90 µM) when compared to parthenolide (EC50 = 6.0) and showed potent antileukemic activity against five primary AML cell lines (EC50 = 0.76-7.3 µM).

Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as cis-constrained analogues of combretastatin A-4.

A series of combretastatin A-4 (CA-4) analogues have been prepared from (Z)-substituted diarylacrylonitriles (1a-1p) obtained in a two-step synthesis from appropriate arylaldehydes and acrylonitriles. The resulting 4,5-disubstituted 2H-1,2,3-triazoles were evaluated for their anti-cancer activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogue 2l exhibited the most potent anti-cancer activity in the screening studies, with GI50 values of <10 nM against almost all the cell lines in the human cancer cell panel and TGI values of <10 nM against cancer cell lines SF-539, MDA-MB-435, OVCAR-3 and A498. Furthermore, in silico docking studies of compounds 2l, 2e and 2h within the active site of tubulin were carried out in order to rationalize the mechanism of the anti-cancer properties of these compounds. From the in silico studies, compound 2e was predicted to have better affinity for the colchicine binding site on tubulin compared to compounds 2l and 2h. Analogue 2e was also evaluated for its anti-cancer activity by colony formation assay against 9LSF rat gliosarcoma cells and afforded an LD50 of 7.5 nM. A cell cycle redistribution assay using analogue 2e was conducted to further understand the mechanism of action of these CA-4 analogues. From this study, analogues 2e and 2l were the most potent anti-cancer agents in this structural class, and were considered lead compounds for further development as anti-cancer drugs.

Three variations in rabbit angiographic stroke models.

PURPOSE: To develop angiographic models of embolic stroke in the rabbit using pre-formed clot or microspheres to model clinical situations ranging from transient ischemic events to severe ischemic stroke. MATERIALS AND METHODS: New Zealand White rabbits (N=151) received angiographic access to the internal carotid artery (ICA) from a femoral approach. Variations of emboli type and quantity of emboli were tested by injection into the ICA. These included fresh clots (1.0-mm length, 3-6h), larger aged clots (4.0-mm length, 3 days), and 2 or 3 insoluble microspheres (700-900 µm). Neurological assessment scores (NAS) were based on motor, sensory, balance, and reflex measures. Rabbits were euthanized at 4, 7, or 24h after embolization, and infarct volume was measured as a percent of total brain volume using 2,3,5-triphenyltetrazolium chloride (TTC). RESULTS: Infarct volume percent at 24 h after stroke was lower for rabbits embolized with fresh clot (0.45±0.14%), compared with aged clot (3.52±1.31%) and insoluble microspheres (3.39±1.04%). Overall NAS (including posterior vessel occlusions) were positively correlated to infarct volume percent measurements in the fresh clot (r=0.50), aged clot (r=0.65) and microsphere (r=0.62) models (p<0.001). CONCLUSION: The three basic angiographic stroke models may be similar to human transient ischemic attacks (TIA) (fresh clot), major strokes that can be thrombolysed (aged clot), or major strokes with insoluble emboli such as atheromata (microspheres). Model selection can be tailored to specific research needs.

Influences of microbubble diameter and ultrasonic parameters on in vitro sonothrombolysis efficacy.

PURPOSE: To quantify the effects of microbubble (MB) size, elasticity, and pulsed ultrasonic parameters on in vitro sonothrombolysis (ultrasound [US]-mediated thrombolysis) efficacy. MATERIALS AND METHODS: Monodispersive MBs with diameters of 1 µm or 3 µm were exposed to pulsed US (1 MHz or 3 MHz) to lyse rabbit blood clots. Sonothrombolysis efficacy (clot mass loss) was measured as functions of MB size and concentration, ultrasonic frequency and intensity, pulse duration (PD), pulse repeat frequency (PRF), and duty factor. RESULTS: Sonothrombolysis at 1 MHz was more effective using 3-µm MBs and at 3 MHz using 1-µm MBs. Sonothrombolysis was more effective at 1 MHz when≥75% of MBs remained intact, especially for 3-µm MBs; improving sonothrombolysis by increasing PRF from 100 Hz to 400 Hz at 3 MHz was associated with increasing 3-µm MB survival. However, 60% of 1-µm MBs were destroyed during maximal sonothrombolysis at 3 MHz, indicating that considerable MB collapse may be required for sonothrombolysis under these conditions. CONCLUSIONS: The ability to control MB size and elasticity permits using a wide range of US parameters (eg, frequency, intensity) to produce desired levels of sonothrombolysis. Comparable, maximal sonothrombolysis efficacy was achieved at 20-fold lower intensity with 3-µm MBs (0.1W/cm(2)) than with 1-µm MBs (2.0W/cm(2)), a potential safety issue for in vivo sonothrombolysis. US parameters that maximized MB survival yielded maximal sonothrombolysis efficacy except with 1-µm MBs at 3MHz where most MBs were destroyed.

Production of uniformly sized serum albumin and dextrose microbubbles.

Uniformly-sized preparations with average microbubble (MB) diameters from 1 to 7 µm were produced reliably by sonicating decafluorobutane-saturated solutions of serum albumin and dextrose. Detailed protocols for producing and size-separating the MBs are presented, along with the effects that changing each production parameter (serum albumin concentration, sonication power, sonication time, etc.) had on MB size distribution and acoustic stability. These protocols can be used to produce MBs for experimental applications or serve as templates for developing new protocols that yield MBs with physical and acoustic properties better suited to specific applications. Size stability and ultrasonic performance quality control tests were developed to assure that successive MB preparations perform identically and to distinguish the physical and acoustic properties of identically sized MBs produced with different serum albumin-dextrose formulations and sonication parameters. MBs can be stored at 5 °C for protracted periods (2 weeks to one year depending on formulation).

Correlation of MRI findings to histology of acetaminophen toxicity in the mouse.

Acetaminophen (APAP) toxicity is responsible for approximately half of all cases of acute liver failure in the United States. The mouse model of APAP toxicity is widely used to examine mechanisms of APAP toxicity. Noninvasive approaches would allow for serial measurements in a single animal to study the effects of experimental interventions on the development and resolution of hepatocellular necrosis. The following study examined the time course of hepatic necrosis using small animal magnetic resonance imaging (MRI) following the administration of 200 mg/kg ip APAP given to B6C3F1 male mice. Mice treated with saline served as controls (CON). Other mice received treatment with the clinical antidote N-acetylcysteine (APAP+NAC). Mouse liver pathology was characterized using T1- and T2-weighted sequences at 2, 4, 8 and 24 h following APAP administration. Standard assays for APAP toxicity [serum alanine aminotransaminase (ALT) levels and hematoxylin and eosin (H&E) staining of liver sections] were examined relative to MRI findings. Overall, T2 sequences had a greater sensitivity for necrosis and hemorrhage than T1 (FLASH) images. Liver injury severity scoring of MR images demonstrated increased scores in the APAP mice at 4, 8 and 24 h compared to the CON mice. APAP+NAC mice had MRI scores similar to the CON mice. Semiquantitative analysis of hepatic hemorrhage strongly correlated with serum ALT. Small animal MRI can be used to monitor the evolution of APAP toxicity over time and to evaluate the response to therapy.

Dodecafluoropentane emulsion decreases infarct volume in a rabbit ischemic stroke model.

PURPOSE: To assess the efficacy of dodecafluoropentane emulsion (DDFPe), a nanodroplet emulsion with significant oxygen transport potential, in decreasing infarct volume in an insoluble-emboli rabbit stroke model. MATERIALS AND METHODS: New Zealand White rabbits (N = 64; weight, 5.1 ± 0.50 kg) underwent angiography and received embolic spheres in occluded internal carotid artery branches. Rabbits were randomly assigned to groups in 4-hour and 7-hour studies. Four-hour groups included control (n = 7, embolized without treatment) and DDFPe treatment 30 minutes before stroke (n = 7), at stroke onset (n = 8), and 30 minutes (n = 5), 1 hour (n = 7), 2 hours (n = 5), or 3 hours after stroke (n = 6). Seven-hour groups included control (n = 6) and DDFPe at 1 hour (n = 8) and 6 hours after stroke (n = 5). DDFPe dose was a 2% weight/volume intravenous injection (0.6 mL/kg) repeated every 90 minutes as time allowed. After euthanasia, infarct volume was determined by vital stains on brain sections. RESULTS: At 4 hours, median infarct volume decreased for all DDFPe treatment times (pretreatment, 0.30% [P = .004]; onset, 0.20% [P = .004]; 30 min, 0.35% [P = .009]; 1 h, 0.30% [P = .01]; 2 h, 0.40% [P = .009]; and 3 h, 0.25% [P = .003]) compared with controls (3.20%). At 7 hours, median infarct volume decreased with treatment at 1 hour (0.25%; P = .007) but not at 6 hours (1.4%; P = .49) compared with controls (2.2%). CONCLUSIONS: Intravenous DDFPe in an animal model decreases infarct volumes and protects brain tissue from ischemia, justifying further investigation.

Successful microbubble sonothrombolysis without tissue-type plasminogen activator in a rabbit model of acute ischemic stroke.

BACKGROUND AND PURPOSE: Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue-type plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with 3 types of MB using a rabbit embolic stroke model. METHODS: New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3-day-old cylindrical clots (0.6 × 4.0 mm). Groups included: (1) control (n=11) embolized without treatment; (2) tPA (n=20); (3) tPA+US (n=10); (4) perflutren lipid MB+US (n=16); (5) albumin 3 µm MB+US (n=8); and (6) tagged albumin 3 µm MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm²) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3 µm MB was 5 × 109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on hematoxylin and eosin sections. RESULTS: Infarct volume percent was lower for rabbits treated with lipid MB+US (1.0%± 0.6%; P=0.013), 3 µm MB+US (0.7% ± 0.9%; P=0.018), and tagged 3 µm MB+US (0.8% ± 0.8%; P=0.019) compared with controls (3.5%± 0.8%). The 3 MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2% ± 0.6% and 1.7%± 0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=nonsignificant). CONCLUSIONS: Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress.

The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer's disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aß) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes-regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.