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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.

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Figure 1: Procedure for implantation of a cerebellar window.
Figure 2: Ultrasonography-based imaging through the cerebellar window.
Figure 3: Cerebellar windows can be applied for the evaluation of indirect surrogates of tumor growth and response to therapies.
Figure 4: Intravital multiphoton microscopy imaging of D283-MED-Gluc medulloblastomas in the cerebellum of mice using a cerebellar window.
Figure 5: OFDI through a cerebellar window.

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Acknowledgements

This study was supported by grants from the US National Cancer Institute (P01-CA 080124, R01-CA163815- and R35-CA197743), the Alex's Lemonade Stand Foundation, and the National Foundation for Cancer Research (to R.K.J.). Support was also provided by the German Research Foundation (Deutsche Forschungsgemeinschaft (DFG) to V.A.), a Jane's Trust Foundation Postdoctoral Fellowship (to M.B.), an Aid for Cancer Research Fellowship (to Z.A.), and Susan G. Komen Foundation Fellowships (to G.S. and G.B.F.).

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Authors

Contributions

V.A., M.B., S.R., D.G.D. and R.K.J. contributed to the concept and design of the study. V.A., M.B., S.R., A.B., N.K., Z.A., G.B.F. and S.C. were responsible for acquisition of the data. V.A., M.B., A.B., N.K., M.S., G.S., G.B.F., L.X., D.F., D.G.D. and R.K.J. contributed to analysis and interpretation of the data. V.A., M.B., S.R., A.B., N.K., M.S., Z.A., G.S., G.B.F., S.C., L.X., D.F., D.G.D. and R.K.J. were involved in drafting of the manuscript and revising it for important intellectual content.

Corresponding author

Correspondence to Rakesh K Jain.

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Competing interests

R.K.J. received consultant fees from Enlight, Ophthotech, Pfizer, SPARC and SynDevRx, owns equity in Enlight, Ophthotech, SynDevRx and XTuit, and serves on the Board of Directors of XTuit and the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund. No funding or reagents from these companies were used in this study.

Integrated supplementary information

Supplementary Figure 1 Tumor cell implantation into the mouse cerebellum.

Orthotopic medulloblastoma model. (A) Schematic of a mouse brain indicating the coordinates for stereotactic implantation of medulloblastoma cells in the cerebellum. (B) Human medulloblastoma (D283-MED, white arrow) in the right hemisphere of the cerebellum of a nude mouse 4 weeks after stereotactic implantation.

Supplementary Figure 2 Reactive gliosis was monitored in cerebellum under window.

A) No primary antibody control IHC with Hematoxylin counterstain reveals intact granular layer in proximity of the window (left for GFAP, right for Iba1). (B) Four representative IHC images of GFAP (left) and Iba1 (right) in “no window” controls, 2 and 10 days post surgery. Both astrocytes and microglia are present in white matter tracts and sparsely in gray matter (scale bar = 200μm). Primary antibodies: mouse anti-GFAP Cat. No. M0761 (DAKO) 1:50 diluted in 5% NGS; rabbit anti-Iba1 Cat. No. 019-19741 (WAKO) 1:500 diluted in 5% NGS. Polymer HRP conjugated secondary antibodies (DAKO) were used (Cat. No. K4011 for rabbit; K4007 for mouse). HRP reaction developed with DAB + substrate reagent from DAKO (Cat. No. K3468).

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Askoxylakis, V., Badeaux, M., Roberge, S. et al. A cerebellar window for intravital imaging of normal and disease states in mice. Nat Protoc 12, 2251–2262 (2017). https://doi.org/10.1038/nprot.2017.101

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