A cerebellar window for intravital imaging of normal and disease states in mice

Abstract

The cerebellum is a prominent part of the vertebrate hindbrain that is critically involved in the regulation of important body functions such as movement coordination, maintenance of balance and posture, and motor control. Here, we describe a cerebellar window that provides access to the mouse cerebellum for intravital imaging, thereby allowing for a detailed characterization of the dynamic processes in this region of the brain. First, the skull overlying the cerebellum is removed, and then the window is applied to the region of interest. Windows may be exchanged depending on the desired imaging modality. This technique has a variety of applications. In the setting of medulloblastoma, spontaneous or orthotopically implanted lesions can be imaged, and tumor morphology and size can be monitored using ultrasonography. Multiphoton laser-scanning microscopy (MPLSM) or optical-frequency-domain imaging (OFDI) can be applied for in vivo visualization and analysis of cellular and vascular structures in a variety of disease states, including malignancies and ataxia telangiectasia. This protocol describes a novel and rapid method for cerebellar window construction that can be set up in under an hour.

Figure 1

Procedure for implantation of a cerebellar window. (a) Remove skin flap, push back the upper neck muscles, and add Gelfoam. (b) Gently drill bone to expose the top of the cerebellum (arrow points to the drill on the surface of the skull). (c) Remove skull piece and keep it moist with Gelfoam (arrows point to the removed skull piece). (d) View of the exposed cerebellum through a light microscope (arrow points to the exposed cerebellum). (e) Place the coverslip (glass or polyethylene) to cover the opening (arrow points to the coverslip). (f) Add saline drops under the coverslip to avoid air bubbles (arrow points to the site of injection under the coverslip). (g) Apply glue at the coverslip–skull bone junction (arrow points to the glue at the coverslip–skull bone junction).

Figure 2

Ultrasonography-based imaging through the cerebellar window. (a) Ultrasonography imaging through a cerebellar window of a human medulloblastoma tumor (D283-MED) stereotactically implanted into the cerebellum of a nude mouse. (b) Ultrasonography imaging of a spontaneous medulloblastoma in a Smo/Smo mouse. (c) Corresponding 3D reconstruction of the tumor from b. Imaging and tumor-volume assessment were performed through a cerebellar window using a polyethylene coverslip and a Vevo 2100 high-frequency ultrasonography device (FujiFilm VisualSonics). All animal procedures were performed according to the guidelines of the Public Health Service Policy on Human Care of Laboratory Animals and in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital. All cell lines used in the experiments depicted were tested both for authenticity (by short tandem-repeat typing) and for mycoplasma contamination, and certified to be both authentic medulloblastoma cell lines and mycoplasma free.

Figure 3

Cerebellar windows can be applied for the evaluation of indirect surrogates of tumor growth and response to therapies. (a) Bioluminescence image of a human medulloblastoma xenograft (D283-MED-Gluc) stereotactically implanted into the cerebellum of a nude mouse. Imaging was performed after retro-orbital injection of a coelenterazine solution (4 mg/kg body weight) using an in vivo imaging system (IVIS Lumina II; Caliper Life Sciences). (b) Correlation between blood Gluc activity and tumor volume for D283-MED-Gluc medulloblastomas growing in the mouse cerebellum, as measured by ultrasonography-based imaging using a cerebellar window (n = 8). (c) Nude mice with medulloblastoma D283-MED-Gluc tumors in the cerebellum were treated with a nonspecific antibody alone or in combination with radiation therapy (2 × 1.8 Gy), and blood Gluc activity was measured over time, as previously described (n = 5–6 per group). Gluc activity was measured as relative light units per s. (d) Survival analysis of mice from c. The survival end point was reached when mice lost > 20% of their body weight or exhibited signs of prolonged distress or neurological impairment. All animal procedures were performed according to the guidelines of the Public Health Service Policy on Human Care of Laboratory Animals and in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital. All cell lines used in the experiments depicted were tested both for authenticity (by short tandem-repeat typing) and for mycoplasma contamination and were certified to be both authentic medulloblastoma cell lines and mycoplasma free. RT, radiation therapy.

Figure 4

Intravital multiphoton microscopy imaging of D283-MED-Gluc medulloblastomas in the cerebellum of mice using a cerebellar window. (a) Intravital multiphoton microscopy images illustrate the medulloblastoma cells (green) in the cerebellum and the tumor vasculature (red). Tumor cells expressing GFP (green) and blood vessels (red) are contrast-enhanced by i.v. injection of tetramethylrhodamine dextran (2,000,000 molecular weight; scale bar, 50 μm). (b) D283-MED-Gluc vessels imaged after i.v. injection of tetramethylrhodamine–BSA (68,000 molecular weight; scale bar, 50 μm) fluorescently labeled red blood cells (RBCs). (c) For quantification of vascular function, fluorescently labeled red blood cells (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD)) were line-scanned (scale bar, 50 μm). (d) The velocity of RBCs was measured by photometric detection of the time required for RBCs to pass between two predetermined positions. (e) Vascular permeability was assessed by quantifying the leakage of tetramethylrhodamine–BSA from the vascular to the extravascular space over a 45-min period. All animal procedures were performed according to the guidelines of the Public Health Service Policy on Human Care of Laboratory Animals and in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital. All cell lines used in the experiments depicted were tested both for authenticity (by short tandem-repeat typing) and for mycoplasma contamination and certified to be both authentic medulloblastoma cell lines and mycoplasma free.

Figure 5

OFDI through a cerebellar window. (a) Normal mouse cerebellum (scale bar, 500 μm). (b) Medulloblastoma (green)—recognized by the combination of the contrast in ‘structure’ and ‘angiography’ parameters—and the vascular network (red)—recognized by the ‘angiography’ OFDI signal—in the mouse cerebellum (scale bar, 500 μm). (c) Serial imaging of the vasculature of medulloblastoma through a cerebellar window. The white arrows indicate the tumor area (scale bar, 100 μm). All animal procedures were performed according to the guidelines of the Public Health Service Policy on Human Care of Laboratory Animals and in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital. All cell lines used in the experiments depicted were tested both for authenticity (by short tandem-repeat typing) and for mycoplasma contamination and certified to be both authentic medulloblastoma cell lines and mycoplasma free.