Expression of mRNAs for IL-1ß, IL-6, IL-10, TNFα, CX3CL1, and TGFß1 Cytokines in the Brain Tissues: Assessment of Contribution of Blood Cells with and without Perfusion.

Cytokines are important regulators of brain function under both normal and pathological conditions. Cytokines can be synthesized by resident cells of the central nervous system (CNS) (vascular endothelium, cells of the blood-brain barrier, parenchymal cells of the CNS) or cells in the lumen of blood vessels, as well as introduced with the bloodstream. The ratio between the quantity of cytokines synthesized in the CNS and those entering it from external sources under various conditions remains poorly understood. In this work, we studied the contribution of mRNAs from non-resident cells to the common pool of cytokine (TNFα, IL-1ß, IL-6, IL-10, CX3CL1, and TGFß1) mRNAs in the rat neocortex, hippocampus, dura matter, pia matter, and choroid plexus. We also evaluated the representation of various populations of resident and non-resident immune cells based on the expression of marker genes (Ncf1, Tbx21, Foxp3, RORγc). The removal of blood by transcardial perfusion led to a decrease in the quantity of the TNFα mRNA in the neocortex and hippocampus and of the IL-1ß, IL-6, and IL-10 mRNAs in the dura mater. The mRNA levels of other cytokines in studied structures were not affected by perfusion. Our findings suggest that mRNAs present in the blood can make a significant contribution to the mRNA levels of some cytokines in the CNS; therefore, preliminary perfusion of brain tissue is a necessary stage of experimental design for correct estimation of mRNA content in the brain.

Activation of microglia associated with lentiviral transduction: A semiautomated method of assessment.

Lentiviral transduction is a powerful tool and widely used in neuroscience research to manipulate gene expression of cells. However, the injection of lentiviral vectors in the brain is not totally benign, it potentially induces focal neuroinflammation. Upon inflammation, microglial cells get activated and can induce major changes in tissue environment, which may interfere with experimental results. In the current study, two weeks after the injection of control viral construction in the dentate gyrus (DG) of rats, an activation of microglia was detected. To access the activation status, we used a fast and accurate method of phenotype detection - measurement of fractal dimension (FD). Microglial morphology is a key indicator of neuroinflammation, therefore FD of microglial cells may serve as a reliable index of inflammation status in the brain. Here we present a detailed description of image processing procedure of images of individual microglial cells. The method allows to preserve the complex structure of microglial cells and their thin processes on the output image, which is important for accurate FD assessment.