Molecular and Cellular Biochemistry

, Volume 453, Issue 1–2, pp 41–51 | Cite as

Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning

  • Manuel Lasch
  • Katharina Nekolla
  • Anna H. Klemm
  • Judith-Irina Buchheim
  • Ulrich Pohl
  • Steffen Dietzel
  • Elisabeth DeindlEmail author


Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.


Arteriogenesis Shear stress Shear rate Two-photon line scanning 



This work was supported by the Fritz Bender Stiftung (ED) and the Friedrich-Baur-Stiftung (AK).

Compliance with ethical standards

Conflict of interest

The authors have declared they have no conflict of interest.


  1. 1.
    Faber JE, Chilian WM, Deindl E, van Royen N, Simons M (2014) A brief etymology of the collateral circulation. Arterioscler Thromb Vasc Biol 34:1854–1859. CrossRefGoogle Scholar
  2. 2.
    Meisner JK, Niu J, Sumer S, Price RJ (2013) Trans-illuminated laser speckle imaging of collateral artery blood flow in ischemic mouse hindlimb. J Biomed Opt 18:096011. CrossRefGoogle Scholar
  3. 3.
    Pipp F, Boehm S, Cai WJ, Adili F, Ziegler B, Karanovic G, Ritter R, Balzer J, Scheler C, Schaper W, S T-R (2004) Elevated fluid shear stress enhances postocclusive collateral artery growth and gene expression in the pig hind limb. Arterioscler Thromb Vasc Biol 24:1664–1668CrossRefGoogle Scholar
  4. 4.
    Drzisga D, Koppl T, Pohl U, Helmig R, Wohlmuth B (2016) Numerical modeling of compensation mechanisms for peripheral arterial stenoses. Comput Biol Med 70:190–201. CrossRefGoogle Scholar
  5. 5.
    Wang QQ, Ping BH, Xu QB, Wang W (2008) Rheological effects of blood in a nonplanar distal end-to-side anastomosis. J Biomech Eng 130:051009. CrossRefGoogle Scholar
  6. 6.
    Errico C, Pierre J, Pezet S, Desailly Y, Lenkei Z, Couture O, Tanter M (2015) Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging. Nature 527:499–502. CrossRefGoogle Scholar
  7. 7.
    Dirnagl U, Villringer A, Einhaupl KM (1992) In-vivo confocal scanning laser microscopy of the cerebral microcirculation. J Microsc 165:147–157CrossRefGoogle Scholar
  8. 8.
    Kleinfeld D, Mitra PP, Helmchen F, Denk W (1998) Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc Natl Acad Sci USA 95:15741–15746CrossRefGoogle Scholar
  9. 9.
    Dietzel S, Pircher J, Nekolla AK, Gull M, Brandli AW, Pohl U, Rehberg M (2014) Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy. PLoS ONE 9:e99615. CrossRefGoogle Scholar
  10. 10.
    Kim TN, Goodwill PW, Chen Y, Conolly SM, Schaffer CB, Liepmann D, Wang RA (2012) Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS ONE 7:e38590. CrossRefGoogle Scholar
  11. 11.
    Santisakultarm TP, Cornelius NR, Nishimura N, Schafer AI, Silver RT, Doerschuk PC, Olbricht WL, Schaffer CB (2012) In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice. Am J Physiol Heart Circ Physiol 302:H1367–H1377. CrossRefGoogle Scholar
  12. 12.
    Shih AY, Friedman B, Drew PJ, Tsai PS, Lyden PD, Kleinfeld D (2009) Active dilation of penetrating arterioles restores red blood cell flux to penumbral neocortex after focal stroke. J Cereb Blood Flow Metab 29:738–751. CrossRefGoogle Scholar
  13. 13.
    Limbourg A, Korff T, Napp LC, Schaper W, Drexler H, Limbourg FP (2009) Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc 4:1737–1746CrossRefGoogle Scholar
  14. 14.
    Pagel JI, Ziegelhoeffer T, Heil M, Fischer S, Fernandez B, Schaper W, Preissner KT, Deindl E (2012) Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation and leukocyte recruitment in vivo. Thromb Haemost 107:562–574. CrossRefGoogle Scholar
  15. 15.
    Burgin-Maunder CS, Nataatmadja M, Vella RK, Fenning AS, Brooks PR, Russell FD (2016) Investigation of long chain omega-3 PUFAs on arterial blood pressure, vascular reactivity and survival in angiotensin II-infused Apolipoprotein E-knockout mice. Clin Exp Pharmacol Physiol 43:174–181. CrossRefGoogle Scholar
  16. 16.
    Rehberg M, Krombach F, Pohl U, Dietzel S (2011) Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy. PLoS ONE 6:e28237. CrossRefGoogle Scholar
  17. 17.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. CrossRefGoogle Scholar
  18. 18.
    Ando J, Yamamoto K (2013) Flow detection and calcium signalling in vascular endothelial cells. Cardiovasc Res 99:260–268. CrossRefGoogle Scholar
  19. 19.
    Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW (1998) Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 82:532–539CrossRefGoogle Scholar
  20. 20.
    Papaioannou TG, Stefanadis C (2005) Vascular wall shear stress: basic principles and methods. Hellenic J Cardiol 46:9–15Google Scholar
  21. 21.
    Koutsiaris AG, Tachmitzi SV, Papavasileiou P, Batis N, Kotoula MG, Giannoukas AD, Tsironi E (2010) Blood velocity pulse quantification in the human conjunctival pre-capillary arterioles. Microvasc Res 80:202–208. CrossRefGoogle Scholar
  22. 22.
    Baker M, Wayland H (1974) On-line volume flow rate and velocity profile measurement for blood in microvessels. Microvasc Res 7:131–143CrossRefGoogle Scholar
  23. 23.
    Tangelder GJ, Slaaf DW, Muijtjens AM, Arts T, oude Egbrink MG, Reneman RS (1986) Velocity profiles of blood platelets and red blood cells flowing in arterioles of the rabbit mesentery. Circ Res 59:505–514CrossRefGoogle Scholar
  24. 24.
    Meier P, Hemingway H, Lansky AJ, Knapp G, Pitt B, Seiler C (2012) The impact of the coronary collateral circulation on mortality: a meta-analysis. Eur Heart J 33:614–621. CrossRefGoogle Scholar
  25. 25.
    Seiler C, Stoller M, Pitt B, Meier P (2013) The human coronary collateral circulation: development and clinical importance. Eur Heart J 34:2674–2682. CrossRefGoogle Scholar
  26. 26.
    Givens C, Tzima E (2016) Endothelial mechanosignaling: does one sensor fit all? Antioxid Redox Signal 25:373–88. CrossRefGoogle Scholar
  27. 27.
    Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA (2005) A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437:426–431. CrossRefGoogle Scholar
  28. 28.
    Lanahan A, Zhang X, Fantin A, Zhuang Z, Rivera-Molina F, Speichinger K, Prahst C, Zhang J, Wang Y, Davis G, Toomre D, Ruhrberg C, Simons M (2013) The neuropilin 1 cytoplasmic domain is required for VEGF-A-dependent arteriogenesis. Dev Cell 25:156–168. CrossRefGoogle Scholar
  29. 29.
    Lautz T, Lasch M, Borgolte J, Troidl K, Pagel JI, Caballero-Martinez A, Kleinert EC, Walzog B, Deindl E (2018) Midkine controls arteriogenesis by regulating the bioavailability of vascular endothelial growth factor A and the expression of nitric oxide synthase 1 and 3. EBioMedicine 27:237–246. CrossRefGoogle Scholar
  30. 30.
    Fischer S, Nishio M, Peters SC, Tschernatsch M, Walberer M, Weidemann S, Heidenreich R, Couraud PO, Weksler BB, Romero IA, Gerriets T, Preissner KT (2009) Signaling mechanism of extracellular RNA in endothelial cells. Faseb J 23:2100–2109CrossRefGoogle Scholar
  31. 31.
    Chillo O, Kleinert EC, Lautz T, Lasch M, Pagel JI, Heun Y, Troidl K, Fischer S, Caballero-Martinez A, Mauer A, Kurz AR, Assmann G, Rehberg M, Kanse SM, Nieswandt B, Walzog B, Reichel CA, Mannell H, Preissner KT, Deindl E (2016) Perivascular Mast cells govern shear stress-induced arteriogenesis by orchestrating leukocyte function. Cell Rep 16:2197–2207. CrossRefGoogle Scholar
  32. 32.
    Chandraratne S, von Bruehl ML, Pagel JI, Stark K, Kleinert E, Konrad I, Farschtschi S, Coletti R, Gartner F, Chillo O, Legate KR, Lorenz M, Rutkowski S, Caballero-Martinez A, Starke R, Tirniceriu A, Pauleikhoff L, Fischer S, Assmann G, Mueller-Hoecker J, Ware J, Nieswandt B, Schaper W, Schulz C, Deindl E, Massberg S (2015) Critical role of platelet glycoprotein ibalpha in arterial remodeling. Arterioscler Thromb Vasc Biol 35:589–597. CrossRefGoogle Scholar
  33. 33.
    Reichel CA, Uhl B, Lerchenberger M, Puhr-Westerheide D, Rehberg M, Liebl J, Khandoga A, Schmalix W, Zahler S, Deindl E, Lorenzl S, Declerck PJ, Kanse S, Krombach F (2011) Urokinase-type plasminogen activator promotes paracellular transmigration of neutrophils via Mac-1, but independently of urokinase-type plasminogen activator receptor. Circulation 124:1848–1859. CrossRefGoogle Scholar
  34. 34.
    Arras M, Ito WD, Scholz D, Winkler B, Schaper J, Schaper W (1998) Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Investig 101:41–50CrossRefGoogle Scholar
  35. 35.
    Badimon L, Badimon JJ, Galvez A, Chesebro JH, Fuster V (1986) Influence of arterial damage and wall shear rate on platelet deposition. Ex vivo study in a swine model. Arteriosclerosis 6:312–320CrossRefGoogle Scholar
  36. 36.
    Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, Sixma JJ (1983) A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 102:522–535Google Scholar
  37. 37.
    Boo YC, Hwang J, Sykes M, Michell BJ, Kemp BE, Lum H, Jo H (2002) Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol 283:H1819–H1828. CrossRefGoogle Scholar
  38. 38.
    Liang S, Slattery MJ, Dong C (2005) Shear stress and shear rate differentially affect the multi-step process of leukocyte-facilitated melanoma adhesion. Exp Cell Res 310:282–292. CrossRefGoogle Scholar
  39. 39.
    Ene-Iordache B, Mosconi L, Antiga L, Bruno S, Anghileri A, Remuzzi G, Remuzzi A (2003) Radial artery remodeling in response to shear stress increase within arteriovenous fistula for hemodialysis access. Endothelium 10:95–102CrossRefGoogle Scholar
  40. 40.
    Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, Busse R, Schaper J, Schaper W (2000) Ultrastructure and molecular histology of rabbit hindlimb collateral artery growth (arteriogenesis). Virchows Arch 436:257–270CrossRefGoogle Scholar
  41. 41.
    Eitenmuller I, Volger O, Kluge A, Troidl K, Barancik M, Cai WJ, Heil M, Pipp F, Fischer S, Horrevoets AJ, Schmitz-Rixen T, Schaper W (2006) The range of adaptation by collateral vessels after femoral artery occlusion. Circ Res 99:656–662CrossRefGoogle Scholar
  42. 42.
    Pohl U, Herlan K, Huang A, Bassenge E (1991) EDRF-mediated shear-induced dilation opposes myogenic vasoconstriction in small rabbit arteries. Am J Physiol 261:H2016–H2023Google Scholar
  43. 43.
    Pohl U, Holtz J, Busse R, Bassenge E (1986) Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8:37–44CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Walter-Brendel-Centre of Experimental MedicineUniversity Hospital, LMU MunichMunichGermany
  2. 2.Core Facility Bioimaging at the Biomedical CenterLMU MunichPlanegg-MartinsriedGermany
  3. 3.Department of Anesthesiology, Laboratory for Stress and ImmunityHospital of the University of the LMU MunichMunichGermany
  4. 4.German Center for Cardiovascular ResearchPartner Site Munich Heart AllianceMunichGermany
  5. 5.Department of Otorhinolaryngology, Head & Neck SurgeryKlinikum der Universität München, Ludwig- Maximilians-Universität MünchenMunichGermany

Personalised recommendations