Resolution of central nervous system astrocytic and endothelial sources of CCL2 gene expression during evolving neuroinflammation

BACKGROUND: The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood–brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further. METHODS: CCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG(35–55) peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells. RESULTS: Both CNS microvessel and parenchymal populations prepared by crude bulk isolation showed up-regulation of CCL2 mRNA following MOG immunization or injection of adjuvants alone. More exact dissection by LCM revealed microvascular endothelial cells and astrocytes to be the specific sources of CCL2 gene induction following MOG immunization, while only astrocytes showed elevated CCL2 mRNA in response to just adjuvants. Astrocytes displayed the greatest degree of stimulation of CCL2 gene expression following EAE induction. CONCLUSIONS: High-precision LCM affirmed both microvascular endothelial cells and astrocytes as the major CNS sources of CCL2 gene expression during EAE. Given the high accessibility of the CNS microvascular endothelium, endothelial-derived CCL2 could prove a viable target for therapeutic intervention in neuroinflammatory disease

Cell-selective Knockout and 3D Confocal Image Analysis Reveals Separate Roles for Astrocyte- and Endothelial-derived CCL2 in Neuroinflammation

Background Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood–brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). Methods Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. Results Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. Conclusions These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease

Crystal growth, structure and thermal properties of noncentrosymmetric single crystals PrCa4O(BO3)3

Noncentrosymmetric praseodymium calcium oxyborate single crystals, PrCa4O(BO3)3 (PrCOB), were grown by the Czochralski technique. The monoclinic unit cell parameters were found to be a = 8.177 Å, b = 16.157 Å, c = 3.629 Å and Z = 2 with space group Cm. Crystal density was measured using the Archimedes method, being on the order of 3.47 g cm-3. Thermal properties of PrCOB were investigated, where the specific heat was found to be 0.63 J g-1 °C-1 at room temperature, increasing to 0.85 J g-1°C-1 at 700°C. The thermal expansion coefficients were measured to be α11 = 7.99, α22 = 4.90 and α33 = 9.46 (10-6/°C), respectively. In addition, thermal diffusivity λ22 and thermal conductivity κ22 as a function of temperature were studied, where λ22 was observed to decrease from 0.89 to 0.58 mm2 s-1, while κ22 was found to maintain the same value, being ∼1.90 W m-1°C-1 over the temperature range of 20-700°C. 2013 The Royal Society of Chemistry

Optimal Design of Compliant Trailing Edge for Shape Changing

AbstractAdaptive wings have long used smooth morphing technique of compliant leading and trailing edge to improve their aerodynamic characteristics. This paper introduces a systematic approach to design compliant structures to carry out required shape changes under distributed pressure loads. In order to minimize the deviation of the deformed shape from the target shape, this method uses MATLAB and ANSYS to optimize the distributed compliant mechanisms by way of the ground approach and genetic algorithm (GA) to remove the elements possessive of very low stresses. In the optimization process, many factors should be considered such as airloads, input displacements, and geometric nonlinearities. Direct search method is used to locally optimize the dimension and input displacement after the GA optimization. The resultant structure could make its shape change from 0 to 9.3 degrees. The experimental data of the model confirms the feasibility of this approach

Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

Recent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB