3D multi-agent models for protein release from PLGA spherical particles with complex inner morphologies

In order to better understand and predict the release of proteins from bioerodible micro- or nanospheres, it is important to know the influences of different initial factors on the release mechanisms. Often though it is difficult to assess what exactly is at the origin of a certain dissolution profile. We propose here a new class of fine-grained multi-agent models built to incorporate increasing complexity, permitting the exploration of the role of different parameters, especially that of the internal morphology of the spheres, in the exhibited release profile. This approach, based on Monte-Carlo (MC) and Cellular Automata (CA) techniques, has permitted the testing of various assumptions and hypotheses about several experimental systems of nanospheres encapsulating proteins. Results have confirmed that this modelling approach has increased the resolution over the complexity involved, opening promising perspectives for future developments, especially complementing in vitro experimentation

In vivo imaging and analysis of cerebrovascular hemodynamic responses and tissue oxygenation in the mouse brain

Published in final edited form as: Nat Protoc. 2018 June ; 13(6): 1377–1402. doi:10.1038/nprot.2018.034.Cerebrovascular dysfunction has an important role in the pathogenesis of multiple brain disorders. Measurement of hemodynamic responses in vivo can be challenging, particularly as techniques are often not described in sufficient detail and vary between laboratories. We present a set of standardized in vivo protocols that describe high-resolution two-photon microscopy and intrinsic optical signal (IOS) imaging to evaluate capillary and arteriolar responses to a stimulus, regional hemodynamic responses, and oxygen delivery to the brain. The protocol also describes how to measure intrinsic NADH fluorescence to understand how blood O2 supply meets the metabolic demands of activated brain tissue, and to perform resting-state absolute oxygen partial pressure (pO2) measurements of brain tissue. These methods can detect cerebrovascular changes at far higher resolution than MRI techniques, although the optical nature of these techniques limits their achievable imaging depths. Each individual procedure requires 1–2 h to complete, with two to three procedures typically performed per animal at a time. These protocols are broadly applicable in studies of cerebrovascular function in healthy and diseased brain in any of the existing mouse models of neurological and vascular disorders. All these procedures can be accomplished by a competent graduate student or experienced technician, except the two-photon measurement of absolute pO2 level, which is better suited to a more experienced, postdoctoral-level researcher.This work was supported by US National Institutes of Health grants R01AG023084, R01NS090904, R01NS034467, R01AG039452, R01NS100459, and P01AG052350 to B.V.Z.; grants R24NS092986, R01EB018464, and R01NS091230 to S.S., S.A.V., and D.A.B.; by funding from the Alzheimer's Association and Cure Alzheimer's fund to B.V.Z.; and by funding from the Fondation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease (ref. no. 16 CVD 05) to B.V.Z. We thank R. Jaswal for helping to create Figure 8. We gratefully acknowledge the feedback, forum posts, and questions from our peers regarding the techniques presented here, which provided the inspiration for the writing of the manuscript. (R01AG023084 - US National Institutes of Health; R01NS090904 - US National Institutes of Health; R01NS034467 - US National Institutes of Health; R01AG039452 - US National Institutes of Health; R01NS100459 - US National Institutes of Health; P01AG052350 - US National Institutes of Health; R24NS092986 - US National Institutes of Health; R01EB018464 - US National Institutes of Health; R01NS091230 - US National Institutes of Health; Alzheimer's Association; Cure Alzheimer's fund; 16 CVD 05 - Fondation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease)https://www.nature.com/articles/nprot.2018.034Accepted manuscrip

FACS isolation of endothelial cells and pericytes from mouse brain microregions

The vasculature is emerging as a key contributor to brain function during neurodevelopment and in mature physiological and pathological states. The brain vasculature itself also exhibits regional heterogeneity, highlighting the need to develop approaches for purifying cells from different microregions. Previous approaches for isolation of endothelial cells and pericytes have predominantly required transgenic mice and large amounts of tissue, and have resulted in impure populations. In addition, the prospective purification of brain pericytes has been complicated by the fact that widely used pericyte markers are also expressed by other cell types in the brain. Here, we describe the detailed procedures for simultaneous isolation of pure populations of endothelial cells and pericytes directly from adult mouse brain microregions using fluorescence-activated cell sorting (FACS) with antibodies against CD31 (endothelial cells) and CD13 (pericytes). This protocol is scalable and takes ∼5 h, including microdissection of the region of interest, enzymatic tissue dissociation, immunostaining, and FACS. This protocol allows the isolation of brain vascular cells from any mouse strain under diverse conditions; these cells can be used for multiple downstream applications, including in vitro and in vivo experiments, and transcriptomic, proteomic, metabolomic, epigenomic, and single-cell analysis