Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

Abstract

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Figure 1.. Cranial window placement, craniotomy, implantation, and imaging.

(A) Cross-section of the anatomical placement of a cranial window. Since the head bar is metal-free, it is possible to perform magnetic resonance imaging or magnetic particle imaging in addition to two-photon intravital microscopy. (B) Overview (upper row) and a detailed view (lower row) of the craniotomy (left), cell implantation (middle), and 2-photon intravital microscopy (right). Upon opening the skull, superficial bleeding occurs (left) and is quickly reabsorbed (middle).

Figure 2.. Computer assisted-design (CAD) of the stage.

CAD of the stage used for in vivo 2-photon intravital microscopy, including a bite bar, head bar stabilization, and screws for height and angle adjustments. The 3D CAD file can be found in the supplement.

Figure 3.. In vivo 2-photon intravital microscopy images.

Images acquired (A) 10 min and (B) 24 h after intravenous nanoparticle administration. While the GBM cells emit fluorescent signals in the red spectrum (left), the nanoparticles in the brain tissue are visualized in green (right). # indicates extravasated NPs, * indicates a blood vessel. Only a limited number of particles have undergone extravasation and are visible in the vicinity of the tumor cells 10 min after NP administration. An abundance of particles is visible in the region of GBM cells, suggesting extravasation 24 h following NP administration. The nanoparticles consist of fluorescein isothiocyanate (FITC) and iron oxide nanoparticles (Ferumoxytol). The scale bars represent 100 μm. 2P-IVM 2-photon intravital microscopy, RFP red fluorescent protein, GBM glioblastoma, FITC fluorescein isothiocyanate, NP nanoparticles.