Intravital Imaging of Neuroimmune Interactions Through a Thinned Skull

Monica Manglani1, Dorian B. McGavern2

1 Medical Scientist Training Program, Pennsylvania State College of Medicine, Hershey, 2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 24.2
DOI:  10.1002/cpim.46
Online Posting Date:  February, 2018
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Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two‐photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue. This protocol provides guidance on how to create a thinned skull window without causing CNS injury. We also describe a highly reproducible method to induce a mild traumatic brain injury using the thinned skull approach. © 2018 by John Wiley & Sons, Inc.

Keywords: intravital imaging; neuroimmune; skull thinning; two‐photon; TBI

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of an Uninflamed Thinned Skull Surgical Window
  • Basic Protocol 2: Generation of a Mild Traumatic Brain Injury (mTBI) Beneath the Thinned Skull Window
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of an Uninflamed Thinned Skull Surgical Window

  • Induction dose of ketamine‐xylazine‐acepromazine (KXA) containing ketamine (85 mg/kg), xylazine (13 mg/kg), and acepromazine (2 mg/kg) in sterile phosphate‐buffered saline (PBS; appendix 2A)
  • Maintenance dose of KXA containing a 1:4 dilution of KXA in sterile phosphate‐buffered saline (PBS; appendix 2A)
  • Ophthalmic lubricant (Lubrifresh PM Ointment, Major Pharmaceuticals, cat, no. 301909)
  • Cyanoacrylate glue (Loctite Super Glue Gel control, cat. no. 234790)
  • Artificial cerebrospinal fluid (aCSF; Harvard Apparatus, cat. no. 597316)
  • Heating pad
  • Scalpel blade (Fine Science Tools, cat no. 10050‐00) or electric shaver
  • Skull‐thinning platform: To stabilize for intravital imaging experiments, our lab uses a custom‐built metal imaging platform stage (15.5 cm × 7.5 cm) with adjustable thumb screws and a metal cranial brace (43 mm × 21 mm with a 5.5 mm diameter center hole) with O‐ring as shown in Figure
  • Surgical forceps and scissors
  • Dissecting microscope with illumination unit (Olympus)
  • Micro‐Drill Kit (Braintree Scientific, cat. no. MD‐1200) with 0.7‐mm burrs for microdrill (Fine Science Tools, cat. no. 19007‐07)
  • Miniature blade, round tip, sharp full radius and sides blade, double bevel, straight (Surgistar, cat. no. 6961)
  • Two‐photon microscope: our lab uses an SP8 two‐photon microscope (Leica) equipped with a Mai Tai HP DeepSee laser (Spectra‐Physics) and an Insight DS laser (Spectra‐Physics), an 12,000‐Hz resonant scanner, a 25×/1.0 NA color‐corrected objective, a quad HyD external detector array, and a custom‐environment chamber
  • Additional reagents and equipment for mouse injection (Donovan & Brown, ) and anesthesia (unit 24.2; Donovan & Brown, )
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Literature Cited

Literature Cited
  Bullen, A., Friedman, R. S., & Krummel, M. F. (2009). Two‐photon imaging of the immune system: A custom technology platform for high‐speed, multicolor tissue imaging of immune responses. Current Topics in Microbiology and Immunology, 334, 1–29. doi: 10.1007/978‐3‐540‐93864‐4_1.
  Cahalan, M. D., & Parker, I. (2008). Choreography of cell motility and interaction dynamics imaged by two‐photon microscopy in lymphoid organs. Annual Review of Immunology, 26, 585–626. doi: 10.1146/annurev.immunol.24.021605.090620.
  Cahalan, M. D., Parker, I., Wei, S. H., & Miller, M. J. (2002). Two‐photon tissue imaging: Seeing the immune system in a fresh light. Nature Reviews Immunology, 2(11)872–880. doi: 10.1038/nri935.
  Carson, M. J., Doose, J. M., Melchior, B., Schmid, C. D., & Ploix, C. C. (2006). CNS immune privilege: Hiding in plain sight. Immunological Reviews, 213, 48–65. doi: 10.1111/j.1600‐065X.2006.00441.x.
  Chaigneau, E., Oheim, M., Audinat, E., & Charpak, S. (2003). Two‐photon imaging of capillary blood flow in olfactory bulb glomeruli. Proceedings of the National Academy of Sciences of the United States of America, 100(22), 13081–13086. doi: 10.1073/pnas.2133652100.
  Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., … Gan, W. B. (2005). ATP mediates rapid microglial response to local brain injury in vivo. Nature Neuroscience, 8(6), 752–758. doi: 10.1038/nn1472.
  Donovan, J., & Brown, P. (2001). Anesthesia. Current Protocols in Immunology, 27, 1.4.1–1.4.5. doi: 10.1002/0471142735.im0104s27.
  Donovan, J., & Brown, P. (2006). Parenteral injections. Current Protocols in Immunology, 73, 1.6.1–1.6.10. doi: 10.1002/0471142735.im0106s73.
  Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E., & Rebane, A. (2011). Two‐photon absorption properties of fluorescent proteins. Nature Methods, 8(5), 393–399. doi: 10.1038/nmeth.1596.
  Fuhrmann, M., Bittner, T., Jung, C. K., Burgold, S., Page, R. M., Mitteregger, G., … Herms, J. (2010). Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease. Nature Neuroscience, 13(4), 411–413. doi: 10.1038/nn.2511.
  Galea, I., Bechmann, I., & Perry, V. H. (2007). What is immune privilege (not)? Trends in Immunology, 28(1), 12–18. doi: 10.1016/
  Gossa, S., Nayak, D., Zinselmeyer, B. H., & McGavern, D. B. (2014). Development of an immunologically tolerated combination of fluorescent proteins for in vivo two‐photon imaging. Scientific Reports, 4, 6664. doi: 10.1038/srep06664.
  Griffin, D. E. (2003). Immune responses to RNA‐virus infections of the CNS. Nature Reviews Immunology, 3(6), 493–502. doi: 10.1038/nri1105.
  Grutzendler, J., Kasthuri, N., & Gan, W. B. (2002). Long‐term dendritic spine stability in the adult cortex. Nature, 420(6917), 812–816. doi: 10.1038/nature01276.
  Herz, J., Zinselmeyer, B. H., & McGavern, D. B. (2012). Two‐photon imaging of microbial immunity in living tissues. Microscopy and Microanalysis, 18(4), 730–741. doi: 10.1017/S1431927612000281.
  Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A., & Littman, D. R. (2000). Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Molecular and Cellular Biology, 20(11), 4106–4114. doi: 10.1128/MCB.20.11.4106‐4114.2000.
  Kang, S. S., & McGavern, D. B. (2009). Inflammation on the mind: Visualizing immunity in the central nervous system. Current Topics in Microbiology and Immunology, 334, 227–263. doi: 10.1007/978‐3‐540‐93864‐4_10.
  Lecoq, J., Parpaleix, A., Roussakis, E., Ducros, M., Goulam Houssen, Y., Vinogradov, S. A., & Charpak, S. (2011). Simultaneous two‐photon imaging of oxygen and blood flow in deep cerebral vessels. Nature Medicine, 17(7), 893–898. doi: 10.1038/nm.2394.
  Louveau, A., Harris, T. H., & Kipnis, J. (2015). Revisiting the mechanisms of CNS immune privilege. Trends in Immunology, 36(10), 569–577. doi: 10.1016/
  Mank, M., Santos, A. F., Direnberger, S., Mrsic‐Flogel, T. D., Hofer, S. B., Stein, V., … Griesbeck, O. (2008). A genetically encoded calcium indicator for chronic in vivo two‐photon imaging. Nature Methods, 5(9), 805–811. doi: 10.1038/nmeth.1243.
  Mulligan, S. J., & MacVicar, B. A. (2004). Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature, 431(7005), 195–199. doi: 10.1038/nature02827.
  Nayak, D., Zinselmeyer, B. H., Corps, K. N., & McGavern, D. B. (2012). In vivo dynamics of innate immune sentinels in the CNS. Intravital, 1(2), 95–106. doi: 10.4161/intv.22823.
  Nimmerjahn, A., Kirchhoff, F., & Helmchen, F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science, 308(5726), 1314–1318. doi: 10.1126/science.1110647.
  Roth, T. L., Nayak, D., Atanasijevic, T., Koretsky, A. P., Latour, L. L., & McGavern, D. B. (2014). Transcranial amelioration of inflammation and cell death after brain injury. Nature, 505(7482), 223–228. doi: 10.1038/nature12808.
  Rua, R., & McGavern, D. B. (2015). Elucidation of monocyte/macrophage dynamics and function by intravital imaging. Journal of Leukocyte Biology, 98(3), 319–332. doi: 10.1189/jlb.4RI0115‐006RR.
  Russo, M. V., & McGavern, D. B. (2015). Immune surveillance of the CNS following infection and injury. Trends in Immunology, 36(10), 637–650. doi: 10.1016/
  Schaffer, C. B., Friedman, B., Nishimura, N., Schroeder, L. F., Tsai, P. S., Ebner, F. F., … Kleinfeld, D. (2006). Two‐photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLOS Biology, 4(2), e22. doi: 10.1371/journal.pbio.0040022.
  Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2(12), 905–909. doi: 10.1038/nmeth819.
  Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B., & Kleinfeld, D. (2012). Two‐photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. Journal of Cerebral Blood Flow and Metabolism, 32(7), 1277–1309. doi: 10.1038/jcbfm.2011.196.
  Sorbara, C., Misgeld, T., & Kerschensteiner, M. (2012). In vivo imaging of the diseased nervous system: An update. Current Pharmaceutical Design, 18(29), 4465–4470. doi: 10.2174/138161212802502279.
  Stosiek, C., Garaschuk, O., Holthoff, K., & Konnerth, A. (2003). In vivo two‐photon calcium imaging of neuronal networks. Proceedings of the National Academy of Sciences of the United States of America, 100(12), 7319–7324. doi: 10.1073/pnas.1232232100.
  Takano, T., Tian, G. F., Peng, W., Lou, N., Libionka, W., Han, X., & Nedergaard, M. (2006). Astrocyte‐mediated control of cerebral blood flow. Nature Neuroscience, 9(2), 260–267. doi: 10.1038/nn1623.
  Tsai, J., Grutzendler, J., Duff, K., & Gan, W. B. (2004). Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nature Neuroscience, 7(11), 1181–1183. doi: 10.1038/nn1335.
  Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., & Axel, R. (2003). Two‐photon calcium imaging reveals an odor‐evoked map of activity in the fly brain. Cell, 112(2), 271–282. doi: 10.1016/S0092‐8674(03)00004‐7.
  Xiong, Y., Mahmood, A., & Chopp, M. (2013). Animal models of traumatic brain injury. Nature Reviews Neuroscience, 14(2), 128–142. doi: 10.1038/nrn3407.
  Xu, H. T., Pan, F., Yang, G., & Gan, W. B. (2007). Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nature Neuroscience, 10(5), 549–551. doi: 10.1038/nn1883.
  Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., & Gan, W. B. (2010). Thinned‐skull cranial window technique for long‐term imaging of the cortex in live mice. Nature Protocols, 5(2), 201–208. doi: 10.1038/nprot.2009.222.
  Zinselmeyer, B. H., Dempster, J., Wokosin, D. L., Cannon, J. J., Pless, R., Parker, I., & Miller, M. J. (2009). Chapter 16: Two‐photon microscopy and multidimensional analysis of cell dynamics. Methods in Enzymology, 461, 349–378. doi: 10.1016/S0076‐6879(09)05416‐0.
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