The blood-cerebrospinal fluid barrier (BCSFB) located in the epithelial cells of the choroid plexus (CP) forms the interface between the cerebrospinal fluid (CSF) and pathogen components circulating in the blood. The CP is also implicated in the passage of peripheral immune signals and circulation of immune cells into the central nervous system. Toll-like receptors (TLRs) are patternrecognition receptors that play a crucial role in the recognition of pathogens and triggering of the innate immune response. In sheep, ten members of the TLR family have been identified and cloned. We used real-time PCR analyses to examine the profiles of TLR mRNA expression in the CP of cerebral ventricles in healthy adult ewes. The transcripts for all ten TLRs except TLR8 were present; however, we observed a high variation in the degree of expression of the TLR5 and TLR1 genes (coefficient of variation: 61% and 46%, respectively) as well as a moderate variation in the expression of the TLR4 (34%), TLR2 (27%) and TLR6 (26%) genes. The TLR9, TLR7, TLR3 and TLR10 genes were the four receptors with relatively invariable expression levels (coefficient of variation: 7%, 8%, 16% and 17%, respectively) across the six adult ewes. The concentration of cortisol in blood collected prior to sacrificing the ewes ranged from 0.18 to 78.9 ng/ml. There was no correlation between cortisol concentration and mRNA expression of any of the examined TLRs. These data suggest that the CP has the potential to sense the presence of many bacterial and viral components and mediate responses for the elimination of invading microorganisms, thereby protecting the brain.

Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood–brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood–cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n = 5) and short day (SD; 8L:16D, n = 5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A ₁₂₀ and VEGF-A ₁₆₄ and our results indicate that VEGF-A ₁₆₄ was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p < 0.05) and NRP-1 mRNA expression (p < 0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.