ex vivo fluorescence microscopy of murine brain tissue sections

<meta charset="utf8"></head><body>Eisenoxid-Nanopartikel für die bildgestützte Therapieverabreichung beim murinen Glioblastom

Glioblastoma multiforme (GBM) is the highest grade of astrocytoma for which there is virtually no cure. Approximately 15,000 people are diagnosed with glioblastoma annually, which has a dismal median survival of about 15 months and a 5-year survival rate of 5%1. In the past decades, there has been marginal improvement in prognosis despite multiple efforts to advance therapeutic options. The current standard of care for GBM includes maximal surgical resection, when feasible, followed by radiotherapy and chemotherapy2. Temozolomide (TMZ), the chemotherapy of choice, was the latest therapy for glioblastoma discovered to show notable clinical efficacy; however, at least 50% of GBM tumors show TMZ resistance3. In spite of this rigorous therapeutic regimen, there is still a significant clinical need for improved glioblastoma therapy.
The development of therapeutics for GBM and other brain-related diseases is significantly hampered by the selective nature of the blood-brain barrier (BBB). The BBB is a physiological barrier comprised of endothelial cells, pericytes, and astrocyte feet-ends, which creates the semi-permeable membrane between the circulatory system and the brain, restricting the free passage of molecules and cells into the brain4. While protective in normal physiology and critical for brain homeostasis, the BBB prevents many therapeutics from reaching the brain, complicating the treatment of GBM. Efforts to enhance the delivery of therapeutics to GBM have led to the development of nanoparticle-based delivery vehicles, focused ultrasound drug delivery enhancement, and receptor-mediated drug delivery5,6.
Nanoparticles have emerged as a promising medium for developing therapeutics for a myriad of diseases, including cancers. The application of nanoparticles for imaging and therapeutic purposes in GBM has been attempted using various nanoparticle constructs7,8. With the focus on delivering drugs to GBM in conjunction with in vivo imaging of the delivery, the proposed approach utilizes magnetic nanoparticles (MN) consisting of an iron oxide core and covered by dextran for stability. The magnetic properties of these nanoparticles afford for their detection by magnetic resonance (MR) imaging, while simple conjugation chemistry to the aminated dextran coating allows for conjugation of therapeutic moieties such as RNA molecules, additional targeting moieties, or imaging moieties (such as Cy5.5 near-infrared optical dye)9,10. In addition to the imaging capabilities, the nano platform is able to extend the half-life of RNA therapeutics by protecting the oligonucleotide from endogenous nucleases, improving therapeutic delivery. Here, the application of this nano platform for in vivo delivery of therapeutic oligonucleotides (termed MN-anti-miR10b) to GBM, monitored by in vivo imaging, is presented. Previously, the ability of this nano platform to accumulate was demonstrated in GBM cells in vitro, causing significant loss of viability of tumor cells11. Prior to performing therapeutic in vivo studies, it is necessary to demonstrate in vivo delivery of this nano platform to GBM tumors in animal models. To achieve this, orthotopic GBM animal models were produced, and intravenous administration of the construct was performed followed by in vivo imaging. Outlined here are the protocols of these studies showing accumulation in the tumor region confirmed by in vivo imaging and ex vivo microscopy.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Innovative Krebstherapien mit Eisenoxid-Nanopartikeln zur Behandlung von Glioblastomen

Nanopartikel-Verabreichung einer Oligonukleotid-Nutzlast in einem Glioblastom-Multiforme-Tiermodell

Our research focuses on the development of cancer therapies using our iron oxide nanoparticle delivery platform. The advantage of our nanoplatform is its ability to cross the blood brain barrier and accumulate in the tumor, delivering therapeutics. The goal is to add to the small arsenal of available therapies for glioblastoma.The blood-brain barrier presents a challenge for the delivery of therapeutics to glioblastoma. Many compounds are unable to cross the barrier, making systemic administration of drugs for glioblastoma treatment near impossible. Currently available systemic treatments for this disease have many off target toxicities.Our nanoplatform overcomes this limitation by helping therapeutic moieties traverse the blood-brain barrier. The delivery of the therapeutic oligonucleotide to the tumor site can be monitored by various imaging modalities, making it a clinically relevant tool. The modular nature of the nanoplatform allows for tailor-made therapies to be developed, offering flexibility to address the vast molecular heterogeneity in glioblastoma subtypes.This precision medicine approach with superior blood-brain barrier penetrance, allows us to expand upon limited therapeutic options.

ex vivo Fluoreszenzmikroskopie von Hirngewebeschnitten von Mäusen

ex-vivo-fluorescence-microscopy-of-murine-brain-tissue-sections

Research

JoVE Journal

Cancer Research

Ex Vivo Fluorescence Microscopy of Murine Brain Tissue Sections

164 Aufrufe. Nehmen Sie zunächst das schockgefrorene Gehirn, das in einer Verbindung mit optimaler Schnitttemperatur gelagert wurde, aus der Lagerung bei 80 Grad Celsius und legen Sie es in die Kryostatkammer. Lassen Sie die Proben auf Kammertemperatur erwärmen. Schneiden Sie das Gehirn mit einem einschneidigen Rasiermesser an der Injektionsstelle koronal auf.Montieren Sie die Probe mit einer kleinen Menge optimaler Schnitttemperatur auf die Probenscheibe. Üben ...

Serie: ex vivo Fluoreszenzmikroskopie von Hirngewebeschnitten von Mäusen

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Forschung

Krebsforschung

MN-anti-miR10b Administration in Murine Glioblastoma Models for Bioluminescence and Fluorescence Imaging 

To begin, weigh the mouse and calculate the volume of magnetic nanoparticle anti-microRNA-10b to administer for a dosage of 20 milligrams iron per kilogram in glioblastoma multiforme. Without introducing bubbles, prepare a 28-gauge syringe with the dose of magnetic nanoparticle anti-microRNA-10b. Sterilize the mouse tail with a 70%isopropanol wipe.After drying, rotate the tail to position the lateral caudal veins at the top of the tail. Then, insert the needle into the tail, and pull back the plunger to establish a vacuum. Continue inserting the needle until the blood enters the syringe.Slowly inject magnetic nanoparticle solution, and retract the needle after complete delivery. Apply pressure to the injection site with gauze until the bleeding stops.

MN-anti-miR10b-Verabreichung in murinen Glioblastommodellen für die Biolumineszenz- und Fluoreszenzbildgebung 

In Vivo and Ex Vivo Imaging for Nanoparticle Tracking in Murine Glioblastoma Models

24 hours after the magnetic nanoparticle, anti-microRNA 10b injection, weigh the mouse and calculate the volume of d-luciferin to administer for a dosage of 150 milligrams per kilogram. Prepare a 28 gauge syringe with the volume protecting the solution from direct light. Once anesthetized, gently scruff the mouse to avoid further injuring the surgical site, and inject the dose of d-luciferin intraperitoneally.Allow the mouse to recover and wait for 10 minutes before imaging. After preparing an in vivo imaging system, or IVIS scanner, place down the black low-fluorescence mat on the imaging stage and configure the nose cone array for imaging. In the software, click imaging wizard.Then, select bioluminescence imaging, followed by open filter. On the next screen, select imaging subject and field of view. Place the anesthetized mouse in a prone position on the IVIS scanner.Click acquire sequence, and using auto exposure settings with a minimum signal threshold of 3000 counts, capture an image for the localization of luciferase-labeled U-251 glioblastoma cells. Next, in the imaging wizard, select fluorescence, followed by filtered pair with epi illumination. In the next screen, select the probe, Cy5.5.Select the imaging subject and field of view. Using auto exposure settings with a minimum signal threshold of 6, 000 counts, capture an image for the localization of magnetic nanoparticles, anti-microRNA 10b. Place the anesthetized mouse prone on the MRI bed.Immobilize and position the head for scanning using a bite bar and ear bars. Install the lubricated rectal temperature probe and ensure respiration and temperature monitoring are functioning. Position the mouse brain coil over the head by fitting the pegs on the mouse bed into the holes on the coil, and tape the coil into place to reduce movement during scanning.Place a small warm water circulating pad over the top of the mouse to maintain body temperature. Move the mouse and imaging bed into position for scanning. In the acquisition software, start the wobble setup step to tune and match the MRI coils.Ensure that the trace is centered and as deep as possible. Acquire a three plane localizer scan of the brain. Using the following parameters, acquire two-dimensional T2 weighted scans to detect the tumor.Acquire a b0 map of the whole brain to calculate a localized shim using the map shim utility. Use three dimensional T2 star-weighted images to visualize the nanoparticles. With the following parameters, acquire a T2 star map for further nanoparticle imaging using the two dimensional T2-weighted image as a reference to position the scan over the tumor.Acquire 10 positive echo images with five milliseconds of echo time spacing. At the experimental endpoint, weigh the mouse, and calculate the volume of d-luciferin to administer for a dosage of 150 milligrams per kilogram. Conduct live bioluminescence imaging 10 minutes post-injection, as previously described.Place the Petri dish with the excised brain from the euthanized mouse in the IVIS scanner. Image the brain with both bioluminescence and fluorescence modalities using the same acquisition settings as the in vivo imaging. The Cy5.5 fluorescence and bioluminescence signals in magnetic nanoparticles anti-microRNA 10b-injected mice showed clear colocalization, indicating delivery of the nanoparticles to the tumor, while control mice showed no fluorescence signal.Ex vivo fluorescence imaging showed the localization of the nanoparticles in major clearance organs, such as the liver and kidneys. A characteristic decrease in T2-weighted MRI signal demonstrates the potential of magnetic nanoparticles, anti-microRNA 10b as an MRI contrast agent to cross the blood-brain barrier and its accumulation in the tumor region.

In vivo und ex vivo Bildgebung für die Verfolgung von Nanopartikeln in murinen Glioblastommodellen

To begin, take the flash frozen brain stored in optimal cutting temperature compound out of 80 degrees Celsius storage, and place it inside the cryostat chamber. Allow the samples to warm to the chamber temperature. Using a single-edged razor, cut the brain coronally at the injection site.Mount the sample onto the specimen disk using a small amount of optimal cutting temperature. Apply ample pressure with a chilled weight inside the chamber for stable mounting of the sample. Move the mounted sample and specimen disk onto the specimen head.Trim the tissue to the desired depth by taking 20-micrometer sections. Once the desired depth is reached, adjust the cryostat to take five to seven micrometer sections of the sample. Then mount the sections onto the pre-labeled glass slide.Fix the sections in the 4%paraformaldehyde for 15 minutes. After fixation, rinse this slide three times with DPBS. Add a 40-microliter drop of mounting medium containing DAPI onto the slide.Carefully place a glass cover slip on top of the medium. Under fluorescence microscopy, visualize the tissue using DAPI and Cy5.5. In ex vivo fluorescence microscopy of tumor tissues, the observed CY 5.5 fluorescent signal confirmed the delivery of magnetic nanoparticles.