Live Confocal Imaging of Zebrafish Notochord Cells Under Mechanical Stress In Vivo

Mechanical stress following injury regulates the quality and speed of wound healing. Improper mechanotransduction can lead to impaired wound healing and scar formation. Vimentin intermediate filaments control fibroblasts’ response to mechanical stress and lack of vimentin makes cells significantly vulnerable to environmental stress. We previously reported the involvement of exosomal vimentin in mediating wound healing. Here we performed in vitro and in vivo experiments to explore the effect of wide-type and vimentin knockout exosomes in accelerating wound healing under osmotic stress condition. Our results showed that osmotic stress increases the size and enhances the release of exosomes. Furthermore, our findings revealed that exosomal vimentin enhances wound healing by protecting fibroblasts against osmotic stress and inhibiting stress-induced apoptosis. These data suggest that exosomes could be considered either as a stress modifier to restore the osmotic balance or as a conveyer of stress to induce osmotic stress-driven conditions.

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DDEL-12. NANOPARTICLE DELIVERY OF DOXORUBICIN FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Neuro-Oncology ◽

10.1093/neuonc/noaa222.047 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii286-iii286

Author(s):

Caitlin Ung ◽

Maria Tsoli ◽

Jie Liu ◽

Domenico Cassano ◽

Dannielle Upton ◽

...

Keyword(s):

Treatment Strategies ◽

Pediatric Brain Tumors ◽

Diffuse Intrinsic Pontine Glioma ◽

Confocal Imaging ◽

Cell Content ◽

Potent Effect ◽

Orthotopic Mouse Model ◽

In Vivo Testing

Abstract DIPGs are the most aggressive pediatric brain tumors. Currently, the only treatment is irradiation but due to its palliative nature patients die within 12 months. Effective delivery of chemotherapy across the blood-brain barrier (BBB) has been a key challenge for the eradication of this disease. We have developed a novel gold nanoparticle functionalised with human serum albumin (Au-NP, 98.8 ±19 nm) for the delivery of doxorubicin. In this study, we evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoparticles (Au-NP-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and Au-NP-Dox (at equimolar concentration) by alamar blue assay. Colony formation assays demonstrated a significantly more potent effect of Au-NP-Dox compared to doxorubicin alone, while the Au-NP had no effect. Furthermore, western blot analysis indicated increased apoptotic markers cleaved Parp, caspase 3/7 and phosphorylated H2AX in Au-NP-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of Au-NP-Dox compared to doxorubicin alone. Treatment of a DIPG orthotopic mouse model with Au-NP-Dox showed no signs of toxicity with stable weights being maintained during treatment. However, in contrast to the above in vitro findings the in vivo study showed no anti-tumor effect possibly due to poor penetration of Au-NP-Dox into the brain. We are currently evaluating whether efficacy can be improved using measures to open the BBB transiently. This study highlights the need for rigorous in vivo testing of new treatment strategies before clinical translation to reduce the risk of administration of ineffective treatments.

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Nanoparticle Delivered Anti-miR-141-3p for Stroke Therapy

Cells ◽

10.3390/cells10051011 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1011

Author(s):

Karishma Dhuri ◽

Rutesh N. Vyas ◽

Leslie Blumenfeld ◽

Rajkumar Verma ◽

Raman Bahal

Keyword(s):

Ischemic Stroke ◽

Nucleic Acid ◽

Double Emulsion ◽

Artery Occlusion ◽

Brain Parenchyma ◽

Confocal Imaging ◽

In Vivo Studies ◽

Stroke Therapy ◽

Systemic Delivery

Ischemic stroke and factors modifying ischemic stroke responses, such as social isolation, contribute to long-term disability worldwide. Several studies demonstrated that the aberrant levels of microRNAs contribute to ischemic stroke injury. In prior studies, we established that miR-141-3p increases after ischemic stroke and post-stroke isolation. Herein, we explored two different anti-miR oligonucleotides; peptide nucleic acid (PNAs) and phosphorothioates (PS) for ischemic stroke therapy. We used US FDA approved biocompatible poly (lactic-co-glycolic acid) (PLGA)-based nanoparticle formulations for delivery. The PNA and PS anti-miRs were encapsulated in PLGA nanoparticles by double emulsion solvent evaporation technique. All the formulated nanoparticles showed uniform morphology, size, distribution, and surface charge density. Nanoparticles also exhibited a controlled nucleic acid release profile for 48 h. Further, we performed in vivo studies in the mouse model of ischemic stroke. Ischemic stroke was induced by transient (60 min) occlusion of middle cerebral artery occlusion followed by a reperfusion for 48 or 72 h. We assessed the blood-brain barrier permeability of PLGA NPs containing fluorophore (TAMRA) anti-miR probe after systemic delivery. Confocal imaging shows uptake of fluorophore tagged anti-miR in the brain parenchyma. Next, we evaluated the therapeutic efficacy after systemic delivery of nanoparticles containing PNA and PS anti-miR-141-3p in mice after stroke. Post-treatment differentially reduced both miR-141-3p levels in brain tissue and infarct injury. We noted PNA-based anti-miR showed superior efficacy compared to PS-based anti-miR. Herein, we successfully established that nanoparticles encapsulating PNA or PS-based anti-miRs-141-3p probes could be used as a potential treatment for ischemic stroke.

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In vivo fibre optic confocal imaging of microvasculature and nerves in the rat vas deferens and colon

Journal of Anatomy ◽

10.1046/j.1469-7580.1998.19240489.x ◽

1998 ◽

Vol 192 (4) ◽

pp. 489-495 ◽

Cited By ~ 23

Author(s):

G. D. PAPWORTH ◽

P. M. DELANEY ◽

L. J. BUSSAU ◽

L. T. VO ◽

R. G. KING

Keyword(s):

Vas Deferens ◽

Rat Vas Deferens ◽

Confocal Imaging ◽

Fibre Optic

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Repetitive tensile stress to rat caudal vertebrae inducing cartilage formation in the spinal ligaments: a possible role of mechanical stress in the development of ossification of the spinal ligaments

Journal of Neurosurgery Spine ◽

10.3171/spi.2006.5.3.234 ◽

2006 ◽

Vol 5 (3) ◽

pp. 234-242 ◽

Cited By ~ 46

Author(s):

Nobuaki Tsukamoto ◽

Takeshi Maeda ◽

Hiromasa Miura ◽

Seiya Jingushi ◽

Akira Hosokawa ◽

...

Keyword(s):

Tensile Stress ◽

Mechanical Stress ◽

Tensile Force ◽

Bone Morphogenetic Protein 2 ◽

Woven Bone ◽

Morphogenetic Protein ◽

Caudal Vertebrae ◽

Spinal Ligaments

Object Mechanical stress has been considered one of the important factors in ossification of the spinal ligaments. According to previous clinical and in vitro studies, the accumulation of tensile stress to these ligaments may be responsible for ligament ossification. To elucidate the relationship between such mechanical stress and the development of ossification of the spinal ligaments, the authors established an animal experimental model in which the in vivo response of the spinal ligaments to direct repetitive tensile loading could be observed. Methods The caudal vertebrae of adult Wistar rats were studied. After creating a novel stimulating apparatus, cyclic tensile force was loaded to rat caudal spinal ligaments at 10 N in 600 to 1800 cycles per day for up to 2 weeks. The morphological responses were then evaluated histologically and immunohistochemically. After the loadings, ectopic cartilaginous formations surrounded by proliferating round cells were observed near the insertion of the spinal ligaments. Several areas of the cartilaginous tissue were accompanied by woven bone. Bone morphogenetic protein–2 expression was clearly observed in the cytoplasm of the proliferating round cells. The histological features of the rat spinal ligaments induced by the tensile loadings resembled those of spinal ligament ossification observed in humans. Conclusions The findings obtained in the present study strongly suggest that repetitive tensile stress to the spinal ligaments is one of the important causes of ligament ossification in the spine.

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Abstract 418: Caveolar Disruption of L-type Calcium Channel and Ryanodine Receptor Facilitates Atrial Ectopy and Arrhythmogenesis in Heart Failure Mice

Circulation Research ◽

10.1161/res.127.suppl_1.418 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Di Lang ◽

Lucas ratajczyk ◽

Leonid Tyan ◽

Daniel Turner ◽

Francisco Alvarado ◽

...

Keyword(s):

Heart Failure ◽

Optical Mapping ◽

Ryanodine Receptors ◽

Tissue Level ◽

Confocal Imaging ◽

Right Atrial ◽

Isolated Atria ◽

The Right

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed in vivo ECG telemetry, in vitro optical mapping and confocal imaging of Ca 2+ transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca 2+ channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca 2+ inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca 2+ releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO, p <0.01 for HF and KO vs WT respectively) especially at the subsarcolemmal space that prolongs CaT-RT (62.2±4.1 ms in WT vs 122.5±12.8 ms in KO, p <0.01). In addition, failing ICR myocytes showed a higher occurrence and size of spontaneous Ca 2+ sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca 2+ release and elevated fibrosis.

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Precise closure of single blood vessels via multiphoton absorption–based photothermolysis

Science Advances ◽

10.1126/sciadv.aan9388 ◽

2019 ◽

Vol 5 (5) ◽

pp. eaan9388 ◽

Cited By ~ 1

Author(s):

Yimei Huang ◽

Zhenguo Wu ◽

Harvey Lui ◽

Jianhua Zhao ◽

Shusen Xie ◽

...

Keyword(s):

Blood Vessels ◽

Real Time ◽

Vascular Diseases ◽

Confocal Imaging ◽

Multiphoton Absorption ◽

Vessel Occlusion ◽

Novel Approach ◽

Vessel Closure ◽

Stroke Models

We report a novel approach to selectively close single blood vessels within tissue using multiphoton absorption–based photothermolysis (multiphoton photothermolysis) without the need of exogenous agents. The treatment process is monitored by in vivo reflectance confocal microscopy in real time. Closure of single targeted vessels of varying sizes ranging from capillaries to venules was demonstrated. We also demonstrated that deeply situated blood vessels could be closed precisely while preserving adjacent overlying superficial blood vessels. In vivo confocal Raman spectroscopy of the treatment sites confirmed vessel closure as being mediated by local coagulative damage. Partial vessel occlusion could be achieved, and it is accompanied by increased intravascular blood cell speed. Multiphoton photothermolysis under real-time reflectance confocal imaging guidance provides a novel precision medicine approach for noninvasive, precise microsurgery treatment of vascular diseases on a per-vessel/per-lesion basis. The method could also be used for building ischemic stroke models for basic biology study.

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Characterization of LAMP1-labeled nondegradative lysosomal and endocytic compartments in neurons

The Journal of Cell Biology ◽

10.1083/jcb.201711083 ◽

2018 ◽

Vol 217 (9) ◽

pp. 3127-3139 ◽

Cited By ~ 63

Author(s):

Xiu-Tang Cheng ◽

Yu-Xiang Xie ◽

Bing Zhou ◽

Ning Huang ◽

Tamar Farfel-Becker ◽

...

Keyword(s):

Motor Neurons ◽

Distribution Patterns ◽

Confocal Imaging ◽

Lysosomal Hydrolases ◽

Differential Distribution ◽

Gradient Fractionation ◽

Endocytic Compartments ◽

Lateral Sclerosis

Despite widespread distribution of LAMP1 and the heterogeneous nature of LAMP1-labeled compartments, LAMP1 is routinely used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomes. In this study, we use immunoelectron microscopy and confocal imaging to provide quantitative analysis of LAMP1 distribution in various autophagic and endolysosomal organelles in neurons. Our study demonstrates that a significant portion of LAMP1-labeled organelles do not contain detectable lysosomal hydrolases including cathepsins D and B and glucocerebrosidase. A bovine serum albumin–gold pulse–chase assay followed by ultrastructural analysis suggests a heterogeneity of degradative capacity in LAMP1-labeled endolysosomal organelles. Gradient fractionation displays differential distribution patterns of LAMP1/2 and cathepsins D/B in neurons. We further reveal that LAMP1 intensity in familial amyotrophic lateral sclerosis–linked motor neurons does not necessarily reflect lysosomal deficits in vivo. Our study suggests that labeling a set of lysosomal hydrolases combined with various endolysosomal markers would be more accurate than simply relying on LAMP1/2 staining to assess neuronal lysosome distribution, trafficking, and functionality under physiological and pathological conditions.

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