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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References
Drew, P.J. et al. Chronic optical access through a polished and reinforced thinned skull. Nat. Methods 7, 981–984 (2010).
Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).
Chow, D.K. et al. Laminar and compartmental regulation of dendritic growth in mature cortex. Nat. Neurosci. 12, 116–118 (2009).
Brown, E.B. et al. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat. Med. 7, 864–868 (2001).
Cabrales, P. & Carvalho, L.J. Intravital microscopy of the mouse brain microcirculation using a closed cranial window. J. Visual. Exp. (45), e2184, http://dx.doi.org/10.3791/2184 (2010).
Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).
Goldey, G.J. et al. Removable cranial windows for long-term imaging in awake mice. Nat. Protoc. 9, 2515–2538 (2014).
Snuderl, M. et al. Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma. Cell 152, 1065–1076 (2013).
Yuan, F. et al. Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows. Cancer Res. 54, 4564–4568 (1994).
Foersch, S. et al. Confocal laser endomicroscopy for diagnosis and histomorphologic imaging of brain tumors in vivo. PLoS One 7, e41760 (2012).
Vakoc, B.J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nat. Med. 15, 1219–1223 (2009).
Kodack, D.P. et al. Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases. Proc. Natl. Acad. Sci. USA 109, E3119–E3127 (2012).
Askoxylakis, V. et al. Preclinical efficacy of Ado-trastuzumab emtansine in the brain microenvironment. J. Natl. Cancer Inst. 108 http://dx.doi.org/10.1093/jnci/djv313 (2016).
Lathia, J.D. et al. Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells. PLoS One 6, e24807 (2011).
Strachan, C.J., Windbergs, M. & Offerhaus, H.L. Pharmaceutical applications of non-linear imaging. Int. J. Pharm. 417, 163–172 (2011).
Yun, S., Tearney, G., de Boer, J., Iftimia, N. & Bouma, B. High-speed optical frequency-domain imaging. Opt. Exp. 11, 2953–2963 (2003).
Tannous, B.A. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat. Protoc. 4, 582–591 (2009).
Bovenberg, M.S., Degeling, M.H. & Tannous, B.A. Enhanced Gaussia luciferase blood assay for monitoring of in vivo biological processes. Anal. Chem. 84, 1189–1192 (2012).
Prajapati, S.I. et al. MicroCT-based virtual histology evaluation of preclinical medulloblastoma. Mol. Imaging Biol. 13, 493–499 (2011).
Suero-Abreu, G.A. et al. In vivo Mn-enhanced MRI for early tumor detection and growth rate analysis in a mouse medulloblastoma model. Neoplasia 16, 993–1006 (2014).
Samano, A.K. et al. Functional evaluation of therapeutic response for a mouse model of medulloblastoma. Transg. Res. 19, 829–840 (2010).
Nelson, A.L. et al. Magnetic resonance imaging of patched heterozygous and xenografted mouse brain tumors. J. Neuro-Oncol. 62, 259–267 (2003).
Jain, R.K. et al. Intravital microscopy of normal and diseased tissues in the mouse. in Live Cell Imaging: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) 435–466 (2005).
Vakoc, B.J., Fukumura, D., Jain, R.K. & Bouma, B.E. Cancer imaging by optical coherence tomography: preclinical progress and clinical potential. Nat. Rev. Cancer 12, 363–368 (2012).
Niermann, K.J. et al. Measuring tumor perfusion in control and treated murine tumors: correlation of microbubble contrast-enhanced sonography to dynamic contrast-enhanced magnetic resonance imaging and fluorodeoxyglucose positron emission tomography. J. Ultrasound Med. 26, 749–756 (2007).
Kamoun, W.S. et al. Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat. Methods 7, 655–660 (2010).
Fukumura, D., Duda, D.G., Munn, L.L. & Jain, R.K. Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models. Microcirculation 17, 206–225 (2010).
Jain, R.K., Munn, L.L. & Fukumura, D. Dissecting tumour pathophysiology using intravital microscopy. Nat. Rev. Cancer 2, 266–276 (2002).
Kloepper, J. et al. Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival. Proc. Natl. Acad. Sci. USA 113, 4476–4481 (2016).
Gillies, R.J., Anderson, A.R., Gatenby, R.A. & Morse, D.L. The biology underlying molecular imaging in oncology: from genome to anatome and back again. Clin. Radiol. 65, 517–521 (2010).
Folarin, A.A., Konerding, M.A., Timonen, J., Nagl, S. & Pedley, R.B. Three-dimensional analysis of tumour vascular corrosion casts using stereoimaging and micro-computed tomography. Microvasc. Res. 80, 89–98 (2010).
Lassau, N. et al. Metastatic renal cell carcinoma treated with sunitinib: early evaluation of treatment response using dynamic contrast-enhanced ultrasonography. Clin. Cancer Res. 16, 1216–1225 (2010).
Walk, E.L., McLaughlin. S., Coad.J. & Weed, S.A. Use of high frequency ultrasound to monitor cervical lymph node alterations in mice. PLoS One 9, e100185 (2014).
Horie, S., Chen, R., Li, L., Mori, S. & Kodama, T. Contrast-enhanced high-frequency ultrasound imaging of early stage liver metastasis in a preclinical mouse model. Cancer Lett. 339, 208–213 (2013).
Sastra, S.A. & Olive, K.P. Quantification of murine pancreatic tumors by high-resolution ultrasound. Methods Mol. Biol. 980, 249–266 (2013).
Li, L. et al. Enhanced sonographic imaging to diagnose lymph node metastasis: importance of blood vessel volume and density. Cancer Res. 73, 2082–2092 (2013).
Xuan, J.W. et al. Functional neoangiogenesis imaging of genetically engineered mouse prostate cancer using three-dimensional power Doppler ultrasound. Cancer Res. 67, 2830–2839 (2007).
Weigert, R., Porat-Shliom, N. & Amornphimoltham, P. Imaging cell biology in live animals: ready for prime time. J. Cell Biol. 201, 969–979 (2013).
Alexander, S., Weigelin, B., Winkler, F. & Friedl, P. Preclinical intravital microscopy of the tumour-stroma interface: invasion, metastasis, and therapy response. Curr. Opin. Cell Biol. 25, 659–671 (2013).
Jain, R.K. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell 26, 605–622 (2014).
Helmlinger, G., Yuan, F., Dellian, M. & Jain, R.K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat. Med. 3, 177–182 (1997).
Kashiwagi, S. et al. Perivascular nitric oxide gradients normalize tumor vasculature. Nat. Med. 14, 255–257 (2008).
Martin, G.R. & Jain, R.K. Noninvasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging microscopy. Cancer Res. 54, 5670–5674 (1994).
Chauhan, V.P. et al. Normalization of tumour blood vessels improves the delivery of nanomedicines in a size-dependent manner. Nat. Nanotechnol. 7, 383–388 (2012).
Ager, E.I. et al. Blockade of MMP14 activity in murine breast carcinomas: implications for macrophages, vessels, and radiotherapy. J. Natl. Cancer Inst. 107, djv017 (2015).
Goel, S. et al. Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression. J. Natl. Cancer Inst. 105, 1188–1201 (2013).
Kirkpatrick, N.D. et al. Video-rate resonant scanning multiphoton microscopy: an emerging technique for intravital imaging of the tumor microenvironment. Intravital 1, 60–68 (2012).
Li, W. et al. Intravital 2-photon imaging of leukocyte trafficking in beating heart. J. Clin. Invest. 122, 2499–2508 (2012).
Bajenoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).
Egeblad, M. et al. Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis. Models Mech. 1, 155–167, discussion 165 (2008).
Sasaki, A., Melder, R.J., Whiteside, T.L., Herbermanm, R.B. & Jain, R.K. Preferential localization of human adherent lymphokine-activated killer cells in tumor microcirculation. J. Natl. Cancer Inst. 83, 433–437 (1991).
Perentes, J.Y. et al. In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts. Nat. Methods 6, 143–145 (2009).
Chauhan, V.P. et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat. Commun. 4, 2516 (2013).
Wei, S.C. et al. Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway. Nat. Cell Biol. 17, 678–688 (2015).
Nakasone, E.S. et al. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell 21, 488–503 (2012).
Condeelis, J. & Segall, J.E. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer 3, 921–930 (2003).
Kedrin, D. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat. Methods 5, 1019–1021 (2008).
Wyckoff, J.B. et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res. 67, 2649–2656 (2007).
Vuong, B. et al. Measuring the optical characteristics of medulloblastoma with optical coherence tomography. Biomed. Opt. Exp. 6, 1487–1501 (2015).
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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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.
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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.
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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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DOI: https://doi.org/10.1038/nprot.2017.101
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