Skip to main content

Advertisement

SpringerLink
Log in

Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy

  • Review
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

A complex and reciprocal communication of cells with each other and with relevant parts of the tissue stroma governs many biological processes in both health and disease. However, in the past, the study of these anatomical and molecular interactions has suffered from a lack of appropriate experimental models. An imaging methodology aimed at changing this should allow intravital display and quantification in an intact non-traumatized organ, imaging over a wide range of time spans including extended periods (i.e., months), many repetitive measurements of the same cell or area to permit the study of the cause and consequence of biological processes, the display of various cell types and their reciprocal interaction with each other in three dimensions, the co-registration of relevant physiological parameters and reporters for selected molecular pathways and as high as possible resolution to visualize sub-cellular structures such as organelles. Remarkably, intravital multiphoton microscopy (in vivo MPLSM) through a chronic cranial window allows us to do all these things, making the brain the inner organ of choice for this technology. Here, we give an overview of the application of in vivo MPLSM to study the choreography of cellular, vascular and molecular interactions in the healthy brain and in neurological diseases. We focus on brain tumor formation, progression and response to therapies. This review further aims at demonstrating that we stand at the beginning of full exploitation of the opportunities provided by this technology and gives clues to future directions that appear most promising.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adelsberger H, Garaschuk O, Konnerth A (2005) Cortical calcium waves in resting newborn mice. Nat Neurosci 8:988–990

    Article  PubMed  CAS  Google Scholar 

  • Attwell D, Buchan AM, Charpak S, Lauritzen M, Macvicar BA, Newman EA (2010) Glial and neuronal control of brain blood flow. Nature 468:232–243

    Article  PubMed  CAS  Google Scholar 

  • Barretto RP, Ko TH, Jung JC, Wang TJ, Capps G, Waters AC, Ziv Y, Attardo A, Recht L, Schnitzer MJ (2011) Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nat Med 17:223–228

    Article  PubMed  CAS  Google Scholar 

  • Bartholomaus I, Kawakami N, Odoardi F, Schlager C, Miljkovic D, Ellwart JW, Klinkert WE, Flugel-Koch C, Issekutz TB, Wekerle H, Flugel A (2009) Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 462:94–98

    Article  PubMed  Google Scholar 

  • Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361:315–325

    Article  PubMed  CAS  Google Scholar 

  • Brown EB, Campbell RB, Tsuzuki Y, Xu L, Carmeliet P, Fukumura D, Jain RK (2001) In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 7:864–868

    Article  PubMed  CAS  Google Scholar 

  • Brown E, McKee T, di Tomaso E, Pluen A, Seed B, Boucher Y, Jain RK (2003) Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat Med 9:796–800

    Article  PubMed  CAS  Google Scholar 

  • Chae SS, Kamoun WS, Farrar CT, Kirkpatrick ND, Niemeyer E, de Graaf AM, Sorensen AG, Munn LL, Jain RK, Fukumura D (2010) Angiopoietin-2 interferes with anti-VEGFR2-induced vessel normalization and survival benefit in mice bearing gliomas. Clin Cancer Res 16:3618–3627

    Article  PubMed  CAS  Google Scholar 

  • Dakin K, Li WH (2006) Infrared-LAMP: two-photon uncaging and imaging of gap junctional communication in three dimensions. Nat Methods 3:959

    Article  PubMed  CAS  Google Scholar 

  • Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8:752–758

    Article  PubMed  CAS  Google Scholar 

  • Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76

    Article  PubMed  CAS  Google Scholar 

  • Ding S, Wang T, Cui W, Haydon PG (2009) Photothrombosis ischemia stimulates a sustained astrocytic Ca2+ signaling in vivo. Glia 57:767–776

    Article  PubMed  Google Scholar 

  • Dittgen T, Nimmerjahn A, Komai S, Licznerski P, Waters J, Margrie TW, Helmchen F, Denk W, Brecht M, Osten P (2004) Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci USA 101:18206–18211

    Article  PubMed  CAS  Google Scholar 

  • Drobizhev M, Makarov NS, Tillo SE, Hughes TE, Rebane A (2011) Two-photon absorption properties of fluorescent proteins. Nat Methods 8:393–399

    Article  PubMed  CAS  Google Scholar 

  • Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51

    Article  PubMed  CAS  Google Scholar 

  • Fuhrmann M, Mitteregger G, Kretzschmar H, Herms J (2007) Dendritic pathology in prion disease starts at the synaptic spine. J Neurosci 27:6224–6233

    Article  PubMed  CAS  Google Scholar 

  • Fuhrmann M, Bittner T, Jung CK, Burgold S, Page RM, Mitteregger G, Haass C, LaFerla FM, Kretzschmar H, Herms J (2010) Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease. Nat Neurosci 13:411–413

    Article  PubMed  CAS  Google Scholar 

  • Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu N, Selig M, Nielsen G, Taksir T, Jain RK, Seed B (1998) Tumor induction of VEGF promoter activity in stromal cells. Cell 94:715–725

    Article  PubMed  CAS  Google Scholar 

  • Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I (2000) Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium 27:97–106

    Article  PubMed  CAS  Google Scholar 

  • Gobel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4:73–79

    Article  PubMed  Google Scholar 

  • Grewe BF, Langer D, Kasper H, Kampa BM, Helmchen F (2010) High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat Methods 7:399–405

    Article  PubMed  CAS  Google Scholar 

  • Haas B, Schipke CG, Peters O, Sohl G, Willecke K, Kettenmann H (2006) Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves. Cereb Cortex 16:237–246

    Article  PubMed  Google Scholar 

  • Hefendehl JK, Wegenast-Braun BM, Liebig C, Eicke D, Milford D, Calhoun ME, Kohsaka S, Eichner M, Jucker M (2011) Long-term in vivo imaging of beta-amyloid plaque appearance and growth in a mouse model of cerebral beta-amyloidosis. J Neurosci 31:624–629

    Article  PubMed  CAS  Google Scholar 

  • Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940

    Article  PubMed  CAS  Google Scholar 

  • Hirase H (2005) A multi-photon window onto neuronal-glial-vascular communication. Trends Neurosci 28:217–219

    Article  PubMed  CAS  Google Scholar 

  • Hirase H, Qian L, Bartho P, Buzsaki G (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol 2:E96

    Article  PubMed  Google Scholar 

  • Hoogland TM, Kuhn B, Gobel W, Huang W, Nakai J, Helmchen F, Flint J, Wang SS (2009) Radially expanding transglial calcium waves in the intact cerebellum. Proc Natl Acad Sci USA 106:3496–3501

    Article  PubMed  CAS  Google Scholar 

  • Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114

    Article  PubMed  CAS  Google Scholar 

  • Kamoun WS, Chae SS, Lacorre DA, Tyrrell JA, Mitre M, Gillissen MA, Fukumura D, Jain RK, Munn LL (2010) Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat Methods 7:655–660

    Article  PubMed  CAS  Google Scholar 

  • Kantevari S, Matsuzaki M, Kanemoto Y, Kasai H, Ellis-Davies GC (2010) Two-color, two-photon uncaging of glutamate and GABA. Nat Methods 7:123–125

    Article  PubMed  CAS  Google Scholar 

  • Kasischke KA, Vishwasrao HD, Fisher PJ, Zipfel WR, Webb WW (2004) Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305:99–103

    Article  PubMed  CAS  Google Scholar 

  • Kasischke KA, Lambert EM, Panepento B, Sun A, Gelbard HA, Burgess RW, Foster TH, Nedergaard M (2011) Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions. J Cereb Blood Flow Metab 31:68–81

    Article  PubMed  CAS  Google Scholar 

  • Kawakami N, Nagerl UV, Odoardi F, Bonhoeffer T, Wekerle H, Flugel A (2005) Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med 201:1805–1814

    Article  PubMed  CAS  Google Scholar 

  • Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE, van Rheenen J (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5:1019–1021

    Article  PubMed  CAS  Google Scholar 

  • Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, Winkler F (2010) Real-time imaging reveals the single steps of brain metastasis formation. Nat Med 16:116–122

    Article  PubMed  CAS  Google Scholar 

  • Koehler RC, Roman RJ, Harder DR (2009) Astrocytes and the regulation of cerebral blood flow. Trends Neurosci 32:160–169

    Article  PubMed  CAS  Google Scholar 

  • Kuchibhotla KV, Lattarulo CR, Hyman BT, Bacskai BJ (2009) Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science 323:1211–1215

    Article  PubMed  CAS  Google Scholar 

  • Kuga N, Sasaki T, Takahara Y, Matsuki N, Ikegaya Y (2011) Large-scale calcium waves traveling through astrocytic networks in vivo. J Neurosci 31:2607–2614

    Article  PubMed  CAS  Google Scholar 

  • Leybaert L, Sanderson MJ (2012) Intercellular Ca(2+) waves: mechanisms and function. Physiol Rev 92:1359–1392

    Article  PubMed  CAS  Google Scholar 

  • Misgeld T, Kerschensteiner M, Bareyre FM, Burgess RW, Lichtman JW (2007) Imaging axonal transport of mitochondria in vivo. Nat Methods 4:559–561

    Article  PubMed  CAS  Google Scholar 

  • Motoike T, Loughna S, Perens E, Roman BL, Liao W, Chau TC, Richardson CD, Kawate T, Kuno J, Weinstein BM et al (2000) Universal GFP reporter for the study of vascular development. Genesis 28:75–81

    Article  PubMed  CAS  Google Scholar 

  • Nagayama S, Zeng S, Xiong W, Fletcher ML, Masurkar AV, Davis DJ, Pieribone VA, Chen WR (2007) In vivo simultaneous tracing and Ca(2+) imaging of local neuronal circuits. Neuron 53:789–803

    Article  PubMed  CAS  Google Scholar 

  • Niesner RA, Hauser AE (2011) Recent advances in dynamic intravital multi-photon microscopy. Cytometry A 79:789–798

    PubMed  Google Scholar 

  • Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37

    Article  PubMed  CAS  Google Scholar 

  • Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318

    Article  PubMed  CAS  Google Scholar 

  • Nolte C, Matyash M, Pivneva T, Schipke CG, Ohlemeyer C, Hanisch UK, Kirchhoff F, Kettenmann H (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33:72–86

    Article  PubMed  CAS  Google Scholar 

  • Sipkins DA, Wei X, Wu JW, Runnels JM, Cote D, Means TK, Luster AD, Scadden DT, Lin CP (2005) In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435:969–973

    Article  PubMed  CAS  Google Scholar 

  • Squirrell JM, Wokosin DL, White JG, Bavister BD (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat Biotechnol 17:763–767

    Article  PubMed  CAS  Google Scholar 

  • Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100:7319–7324

    Article  PubMed  CAS  Google Scholar 

  • Svoboda K, Tank DW, Denk W (1996) Direct measurement of coupling between dendritic spines and shafts. Science 272:716–719

    Article  PubMed  CAS  Google Scholar 

  • Takano T, Han X, Deane R, Zlokovic B, Nedergaard M (2007a) Two-photon imaging of astrocytic Ca2+ signaling and the microvasculature in experimental mice models of Alzheimer’s disease. Ann N Y Acad Sci 1097:40–50

    Article  PubMed  CAS  Google Scholar 

  • Takano T, Tian GF, Peng W, Lou N, Lovatt D, Hansen AJ, Kasischke KA, Nedergaard M (2007b) Cortical spreading depression causes and coincides with tissue hypoxia. Nat Neurosci 10:754–762

    Article  PubMed  CAS  Google Scholar 

  • Tantama M, Hung YP, Yellen G (2011) Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. J Am Chem Soc 133:10034–10037

    Article  PubMed  CAS  Google Scholar 

  • Tatenhorst L, Puttmann S, Senner V, Paulus W (2005) Genes associated with fast glioma cell migration in vitro and in vivo. Brain Pathol 15:46–54

    Article  PubMed  CAS  Google Scholar 

  • Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jain RK (2004) Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 64:3731–3736

    Article  PubMed  CAS  Google Scholar 

  • von Baumgarten L, Brucker D, Tirniceru A, Kienast Y, Grau S, Burgold S, Herms J, Winkler F (2011) Bevacizumab has differential and dose-dependent effects on glioma blood vessels and tumor cells. Clin Cancer Res 17:6192–6205

    Article  Google Scholar 

  • Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9:816–823

    Article  PubMed  CAS  Google Scholar 

  • Winkler F, Kozin SV, Tong RT, Chae SS, Booth MF, Garkavtsev I, Xu L, Hicklin DJ, Fukumura D, di Tomaso E et al (2004) Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell 6:553–563

    PubMed  CAS  Google Scholar 

  • Winkler F, Kienast Y, Fuhrmann M, von Baumgarten L, Burgold S, Mitteregger G, Kretzschmar H, Herms J (2009) Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis. Glia 57:1306–1315

    Article  PubMed  Google Scholar 

  • Winship IR, Plaa N, Murphy TH (2007) Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo. J Neurosci 27:6268–6272

    Article  PubMed  CAS  Google Scholar 

  • Wood S (1958) Pathogenesis of metastasis formation observed in vivo in the rabbit ear chamber. AMA Arch Pathol 66:550–568

    PubMed  Google Scholar 

  • Xu HT, Pan F, Yang G, Gan WB (2007) Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci 10:549–551

    Article  PubMed  CAS  Google Scholar 

  • Yamauchi K, Yang M, Jiang P, Xu M, Yamamoto N, Tsuchiya H, Tomita K, Moossa AR, Bouvet M, Hoffman RM (2006) Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res 66:4208–4214

    Article  PubMed  CAS  Google Scholar 

  • Yuan F, Salehi HA, Boucher Y, Vasthare US, Tuma RF, Jain RK (1994) Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows. Cancer Res 54:4564–4568

    PubMed  CAS  Google Scholar 

  • Yuan F, Chen Y, Dellian M, Safabakhsh N, Ferrara N, Jain RK (1996) Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci USA 93:14765–14770

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Neurooncology, Neurology Clinic and National Center for Tumor Diseases, University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Matthias Osswald & Frank Winkler

Authors
  1. Matthias Osswald
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Winkler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frank Winkler.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Timecourse (1 h 5 min) of the dynamic microglial reaction after laser-induced cell death of a single microglial cell; 40 subsequent z-stacks of 36 slices, scaling 1.5 μm, are shown in 3D view over time. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 1. (AVI 1212 kb)

Three-dimensional reconstruction and simulation (Imaris Bitplane) of a melanoma brain metastasis (green cerebral blood vessels, red melanoma cells); 500×500×400 μm. M. Osswald, unpublished. For video sequence, see Supplementary video 3. (AVI 63296 kb)

Micro- and astroglial cells can be tracked over time (26 min). Whereas the astrocytes remain in a stable network with stable protrusions foming one part of the blood-brain barrier, the microglia patrol the shown brain region. M. Osswald, unpublished. Bar 15 μm. For video sequence, see Supplementary video 4. (AVI 1210 kb)

Supplementary video 4

Astrocytic calcium imaging by cortical application of fluo-4 AM. Changes in intracellular [Ca] are reflected by the change in fluorescent intensity. Thin astrocytic processes can be seen during astrocytic calcium waves (the first three waves from the left evolving to the right side, one wave from the right evolving to the left side). M. Osswald, unpublished. (AVI 5033 kb)

Imaging of tumor cell migration and proliferation in vivo. Lewis lung cancer cells were double-labeled with dsRed (cytoplasm) and GFP (nuclei, H2B). Because of the labeling of the nuclei, single cells can easily be tracked over time. A mitosis can also be seen. M. Osswald, unpublished. Bar 30 μm. (AVI 545 kb)

Glioma-blood-vessel interaction. Video (10 min) of a 3D stack showing a glioma cell (green), which ties up a twisted and angiogenic deformed blood vessel (red). The black discs that can be seen in the blood vessel are erythrocytes and show a highly variable intravascular velocity, depending on the vascular constriction of the tumor cell. M. Osswald, unpublished. Bar 15 μm. (AVI 1091 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osswald, M., Winkler, F. Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy. Cell Tissue Res 352, 149–159 (2013). https://doi.org/10.1007/s00441-013-1580-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-013-1580-3

Keywords

Search

Navigation