Macrophage mediated mesoscale brain mechanical homeostasis in vivo

Introduction:: Mechanical phenotypes have been recognized as one of critical factors for tissue and organ development during morphogenesis. Tissues are active materials where epithelial turnover, immune surveillance and remodeling of stromal cells all regulate form and function. Here, we addressed two outstanding questions: a) how do we reconcile methods that probe mechanical properties at different temporal scales and b) the role of stromal dynamics in regulating normal tissue homeostasis. Here, we demonstrate that the brain tissue mechanical properties are dependent on both the structural presence of the macrophages and the colony stimulating factor 1 receptor (CSF1R) that facilitates stromal remodeling. The mechanical homeostasis was modulated by both acute and permanent alterations of macrophage dynamics. We apply optical based techniques of different spatio-temporal characteristics in a model that recapitulates spatial anisotropy and temporal evolution of mechanical properties in native intact tissues. This enables us to link viscoelastic properties of tissue to tensional homeostasis in a living animal. The zebrafish has long been utilized as a model organism as many organs are conserved in mammalian counterparts. Similar to mammals, zebrafish microglia development also depends on CSF1R. Moreover, the transparency of larval zebrafish allows for non-invasive optical techniques. Using a suite of tools where we either chemo- genetically or chemically ablate stromal constituents, we probed the role of mechanical mediated tissue remodeling in larval zebrafish.


Materials and Methods::

To measure the mechanical properties of the developing brain from 3 - 4dpf, we employed optical trap based active microrheology (OT-AMR) (Figure 1a). First, we introduced 1 micron-diameter polystyrene spheres directly into the brain parenchyma at 2 days post fertilization (2dpf). Using these spheres as mechanical probes, we applied a multiplexed-sinusoidal oscillation based on ~nm amplitude to each bead over a range of frequencies from 7Hz to 15kHz. The displacements of the bead are simultaneously tracked via back focal plane interferometry. Quantitation of the local viscoelasticity, G*(w) at that bead position is achieved by correlating the trap and bead positions.



To complement OT-AMR data, Brillouin microscopy (BM) was used since it is a purely non-invasive technique, based on the intrinsic inelastic scattering due to incident photon-phonon interactions in materials, whose spectral characterization provides information correlated with the underlying mechanical properties (Figure 1b). Formulaically, the frequency shift (v_B) of Brillouin scattered photons (on the order of GHz) is proportional to the longitudinal elastic modulus (M’) of samples. Thus, stiffer samples would result in higher frequency shifts.



After validating the correlation between the two modalities, BM was further utilized to investigate the role of Myosin II on brain parenchyma using Blebbistatin. Moreover, we used various macrophage depletion methods ranging from nanoliposomes encapsulating clodronate to CSF1R inhibitor small drug to probe the macrophage contribution on brain mechanical homeostasis along with null mutation of CSF1R zebrafish. Neutral red staining was used to quantify macrophages at brain over the development and upon drug perturbations.




Results, Conclusions, and Discussions:: We show that brain tissue is mechanically heterogenous as a function of region of the brain and age (Figure 1d). At 3dpf, the forebrain is more rigid than the mid and hindbrain. As the brain develops, all regions exhibit an increase in the complex modulus where the hindbrain showing the greatest increase (Figure 1f). The normalized complex modulus data from whole frequency range by 3 dpf hindbrain shows that each brain regions has distinct mechanical property with statistical significance based on paired t-test (Figure 1d,f).



OT-AMR offers the advantage of quantifying absolute values of viscoelasticity. However, the technique necessitates the introduction of probes, that if introduced in sufficient numbers can perturb normal physiology. Mechanical phenotypes of large tissues can only be obtained by averaging across many samples assuming random bead distribution. Thus, assessing the viscoelasticity of an individual fish is difficult. Then, non-invasive BM was employed to check the correlation between OT-AMR. We determined that Brillouin shifts were comparable in detecting mechanical heterogeneity of brain regions at 3-4dpf (Figure 1c-f). Furthermore, the trends in stiffness at 3-4 dpf based on Brillouin microscopy are the same as those measured using OT-AMR (Figure 1c-f). Blebbistain treatment also induced brain parenchyma softening based on lower frequency shift compared to vehicle control (Figure 1g-h).



Over the development, macrophages at brain increase with increasing stiffness of brain parenchyma based on neutral red staining (Figure 1i). Furthermore, macrophages numbers decreased in fish treated with Blebbistatin compared to vehicle control (Figure 1j). To confirm that the presence of macrophages can directly perturb mechanical properties of the brain, we ablated macrophages using various methods such liposomes encapsulating, chemical ablation, and CSF1R inhibitor. These macrophage-specific ablations caused softening of whole brain parenchyma (Data not shown here).



Here, we identified macrophages as a key factor in brain tissue homeostasis. Mechanical properties are dependent on both the structural presence of the macrophages and the receptor that facilitates stromal remodeling. The mechanical homeostasis was modulated by both acute and permanent alterations of macrophage dynamics. Although our results are obtained using larval fish, these observations may have consequences for adult tissues.


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