PD Dr. Gabriele BixelScientific Administrator
Intravital two-photon imaging of the bone marrow microvasculature
Bone marrow (BM) is the principle site of postnatal haematopoiesis and requires a specialized microenvironment in the bone, the haematopoietic niche, which controls the maintenance and self-renewal of haematopoietic stem cells. Deeper insights into the functional structures of the BM and the dynamics of haematopoietic stem and progenitor cell homing strongly depend on a better understanding of the blood vessel microarchitecture and blood flow dynamics in the various types of BM vessels.
The functional organization and blood flow profile in the BM microvasculature have remained poorly understood due to technical challenges associated with in vivo high-resolution imaging in the intact bone. The key advantages of intravital two-photon imaging of the calvarium through a chronic cranial window are, besides its minimal invasiveness, the possibility of reaching high spatial and temporal resolution with imaging depths up to 300 µm, allowing the analysis of the dynamic cellular behavior in the BM compartment.
We perform direct in vivo two-photon measurements of blood flow dynamics and red blood cell (RBC) velocities at the level of individual arterial and sinusoidal vessels by capturing the motion of RBCs. Real-time imaging of multiple short vessel segments visualize the highly variable RBC flow pattern at cellular resolution, showing strong temporal and spatial fluctuations. In addition, we use repetitive center line scans to acquire additional information on blood flow velocities and flow densities in multiple vessel segments. Arterial vessels with rapid flow and laminar flow pattern connect to a complex interconnecting network of sinusoidal vessels. In these sinusoidal vessels, blood flow slows down considerably and shows a turbulent behavior. Among sinusoidal capillaries, few segments were identified with very barely any RBC flow. These BM regions are likely to be areas of low oxygen tension due to low perfusion with oxygen carrying RBCs.
Goswami D., Maerz S., Li Y.-T, Artz A., Schaefer K., Seelige R., Pacheco-Blanco M., Jing D., Bixel M.G., Araki M., Araki K., Yamamura K., Vestweber D. (2017) Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs. Blood, accepted.
Langen U.H., Pitulescu M.E., Kim J.M., Enriquez-Gasca R., Sivaraj K.K., Kusumbe A.P., Singh A., Di Russo J., Bixel M.G., Zhou B., Sorokin L., Vaquerizas J.M., Adams R.H. (2017) Cell–matrix signals specify bone endothelial cells during developmental osteogenesis. Nat. Cell Biol. DOI:10.1038/ncb3476
Bixel M.G.1), Kusumbe A.P., Ramasamy S.K., Sivaraj K.K., Butz S., Vestweber D. and Adams R.H.1) (2017) Flow dynamics and HSPC homing in bone marrow microvessels. Cell Reports, DOI: 10.1016/j.celrep.2017.01.042
1) Shared corresponding author
Ramasamy S.K., Kusumbe A.P., Schiller M., Zeuschner D., Bixel M.G., Milia C., Gamrekelashvili J., Limbourg A., Medvinsky A., Santoro M.M., Limbourg F.P., Adams R.H. (2016) Blood flow controls bone vascular function and osteogenesis. Nat. Commun. 7:13601
Bixel M.G.1), Fretham S.J. and Aschner M1). (2015) High-resolution multi-photon imaging of morphological structures of caenorhabditis elegans. Curr Protoc Toxicol 64, 11.19.1-11.19.11
1) Shared corresponding author
Wang L., Benedito R., Bixel M.G., Zeuschner D., Stehling M., Sävendahl L., Breier G., Haigh J.J., Kiefer F. and Adams R.H. (2012) Identification of clonally expanding haematopoietic compartment in bone marrow. EMBO J 32, 219-230
Bixel M.G., Li H., Petri B., Khandoga A.G., Khandoga A., Zarbock A., Wolburg-Buchholz K., Wolburg H., Sorokin L., Zeuschner D., Maerz S., Butz S., Krombach F. and Vestweber D. (2010) CD99 and CD99L2 act at the same site as, but indepen-dently of, PECAM-1 during leukocyte diapedesis. Blood 116, 1172-1184
Bixel M.G. and Adams R.H. (2008) Master and commander: continued expression of Prox1 prevents the dedifferentiation of lymphatic endothelial cells. Genes Dev. 22, 3232-5
Nottebaum A.F., Cagna G. Winderlich M., Gamp A.C., Linnepe R., Polaschegg C., Filippova K., Lyck R., Engelhardt B., Kamenyeva O., Bixel M.G., Butz S. and Vestweber D. (2008) VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF. J. Ex. Med. 205, 2929-2945
Yakubenia S., Frommhold D., Schölch D., Hellbusch C.C., Körner C., Petri B., Jones C., Ipe U., Bixel M.G., Krempien R., Sperandio M. and Wild M.K. (2008) Leukocyte trafficking in a mouse model for leukocyte adhesion deficiency II/congenital disorder of glycosylation IIc. Blood.112, 1472-81
Frommhold D., Ludwig A., Bixel M.G., Zarbock A., Babushkina I., Lange-Sperandio B., Ellies L.G., Marth J.D., Beck-Sickinger A.G., Linderkamp O., Vestweber D., Ley K. and Sperandio M. (2008) Sialyltransferase ST3Gal-IV controls CXCR2 mediated firm leukocyte arrest during inflammation. J Exp Med. 205,1435-1446
van Wanrooij E.J.A., de Vos P., Bixel M.G., Vestweber D., van Berkel T.J.C. and Kuiper J. (2008) Vaccination against CD99 inhibits atherogenesis in LDL receptor deficient mice. Cardiovasc. Res. 78, 590-596
Bixel M.G., Petri B., Khandoga A.G., Khandoga A., Wolburg-Buchholz K., Wolburg H., März S., Krombach F. and Vestweber D. (2007) A CD99-related antigen on endothelial cells mediates neutrophil, but not lymphocyte extravasation in vivo. Blood 109, 5327-5336
Wethmar K., Helmus Y., Lühn K., Jones C., Laskowsak A., Varga G., Grabbe S., Lyck R., Engelhardt B., Bixel M.G., Butz S., Loser K., Beissert S., Ipe U., Vestweber D. and Wild M.K. (2006) Migration of immature mouse DC across resting endothelium is mediated by ICAM-2 but independent of β2-integrins and murine DC-SIGN homologues. Eur. J. Immunol. 36, 2781-2794
Bixel G., Kloep S., Butz S., Petri B., Engelhardt B. and Vestweber D. (2004) Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood 104, 3205-3213