ARTICLE Changes induced by hyperosmotic mannitol in cerebral endothelial cells: an atomic force microscopic study Zolta ´n Ba ´lint Æ Istva ´n A. Krizbai Æ Imola Wilhelm Æ Attila E. Farkas Æ A ´ rpa ´d Pa ´ rducz Æ Zsolt Szegletes Æ Gyo ¨ rgy Va ´ro ´ Received: 18 May 2006 / Revised: 20 September 2006 / Accepted: 10 October 2006 / Published online: 8 November 2006 Ó EBSA 2006 Abstract Understanding the reaction of living cells in response to different extracellular stimuli, such as hyperosmotic stress, is of primordial importance. Mannitol, a cell-impermeable non-toxic alcohol, has been used successfully for reversible opening of the blood–brain barrier in hyperosmotic concentrations. In this study we analyzed the effect of hyperosmotic mannitol on the shape and surface structure of living cerebral endothelial cells by atomic force microscope imaging technique. Addition of clinically relevant concentrations of mannitol to the culture medium of the confluent cells induced a decrease of about 40% in the observed height of the cells. This change was consistent both at the nuclear and peripheral region of the cells. After mannitol treatment even a close examination of the contact surface between the cells did not reveal gap between them. We could observe the appearance of surface protrusions of about 100 nm. By force measurements the elasticity of the cells were estimated. While the Young’s modulus of the control cells appeared to be 8.04 ± 0.12 kPa, for the mannitol- treated cells it decreased to an estimated value of 0.93 ± 0.04 kPa which points to large structural chan- ges inside the cell. Keywords Blood–brain barrier  Cell imaging  Cytoskeleton  Force measurement  Young’s modulus Introduction Since the invention of the atomic force microscope (AFM) in 1986 (Binnig et al. 1986), it became a very important tool in the field of biology (Butt et al. 1990; Engel 1991). Besides the classical methods developed for electron microscopy, such as cell fixation with glu- taraldehyde or paraformaldehyde (Moloney et al. 2004), the study of the living cells became possible (Pesen and Hoh 2005a, b; Dufrene 2003). The use of AFM opened the possibility to study directly the effect of extracellular stimuli and the action of different drugs on living cells. Besides investigations of the surface and submem- branous structures of living cells, AFM proved to be a useful tool in the study of spatial and temporal changes of the mechanical properties of different cell types (Hassan et al. 1998; Vinckier and Semenza 1998; Mathur et al. 2001; Sato et al. 2004). Furthermore, different aspects of cellular function such as cell growth on different surfaces (Chung et al. 2003; Domke et al. 2000), volume changes induced by Ca 2+ depletion (Quist et al. 2000) or drug administration (Rotsch and Radmacher 2000) have been studied as well. At a higher resolution AFM is a unique imaging tool for visualizing the cytoskeletal organization of the cells (Le Grimellec et al. 1998; Mahaffy et al. 2004; Berdyyeva et al. 2005; Pesen and Hoh 2005a, b; Sharma et al. 2005). Force measurements on the surface of the cells have revealed the elastic properties of different cells and cell structures (Vinckier and Semenza 1998; Sato et al. 2004; Wojcikiewicz et al. 2004; Sharma et al. 2005). Force measurements on the surface of a membrane-bound protein, with chemically coated Z. Ba ´lint  I. A. Krizbai  I. Wilhelm  A. E. Farkas  A ´ . Pa ´rducz  Z. Szegletes  G. Va ´ro ´(&) Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvari krt 62, Szeged 6726, Hungary e-mail: varo@nucleus.szbk.u-szeged.hu 123 Eur Biophys J (2007) 36:113–120 DOI 10.1007/s00249-006-0112-4