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The study was designed to examine whether the administration of neostigmine (0.5 mg/animal), a peripheral inhibitor of acetylcholinesterase (AChE), during an immune/inflammatory challenge provoked by intravenous injection of bacterial endotoxin—lipopolysaccharide (LPS; 400 ng/kg)—attenuates the synthesis of proinflammatory cytokines in the ovine preoptic area (POA), the hypothalamic structure playing an essential role in the control of the reproduction process, and in the choroid plexus (CP), a multifunctional organ sited at the interface between the blood and cerebrospinal fluid in the ewe. Neostigmine suppressed (p < 0.05) LPS-stimulated synthesis of cytokines such as interleukin- (IL-) 1β, IL-6, and tumor necrosis factor (TNF) α in the POA, and this effect was similar to that induced by the treatment with systemic AChE inhibitor—donepezil (2.5 mg/animal). On the other hand, both AChE inhibitors did not influence the gene expression of these cytokines and their corresponding receptors in the CP. It was found that this structure seems to not express the neuronal acetylcholine (ACh) receptor subunit alpha-7, required for anti-inflammatory action of ACh. The mechanism of action involves inhibition of the proinflammatory cytokine synthesis on the periphery as well as inhibition of their de novo synthesis rather in brain microvessels and not in the CP. In conclusion, it is suggested that the AChE inhibitors incapable of reaching brain parenchyma might be used in the treatment of neuroinflammatory processes induced by peripheral inflammation.

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-A120 and VEGF-A164 and our results indicate that VEGF-A164 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.

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.

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin—lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1β) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer (IL6ST) and TNF receptor superfamily member 1A (TNFRSF1) in the hypothalamus and IL-1 type II receptor (IL1R2) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.

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Background: Interleukin-1β (IL-1β) is important mediator of inflammatory-induced suppression of reproductive axis at the hypothalamic level. At the beginning of inflammation, the main source of cytokines in the cerebrospinal fluid (CSF) is peripheral circulation, while over time, cytokines produced in the brain are more important. Melatonin has been shown to decrease pro-inflammatory cytokines concentration in the brain. In ewes, melatonin is used to advance the onset of a breading season. Little is known about CSF concentration of IL-1β in ewes and its correlation with plasma during inflammation as well as melatonin action on the concentration of IL-1β in blood plasma and the CSF, and brain barriers permeability in early stage of lipopolysaccharide (LPS)-induced inflammation. Methods: Systemic inflammation was induced through LPS administration in melatonin-and sham-implanted ewes. Blood and CSF samples were collected before and after LPS administration and IL-1β and albumin concentration were measured. To assess the functions of brain barriers albumin quotient (QAlb) was used. Expression of IL-1β (Il1B) and its receptor type I (Il1r1) and type II (Il1r2) and matrix metalloproteinase (Mmp) 3 and 9 was evaluated in the choroid plexus (CP). Results: Before LPS administration, IL-1β was on the level of 62.0 ± 29.7 pg/mL and 66.4 ± 32.1 pg/mL in plasma and 26.2 ± 5.4 pg/mL and 21.3 ± 8.7 pg/mL in the CSF in sham-and melatonin-implanted group, respectively. Following LPS it increased to 159.3 ± 53.1 pg/mL and 197.8 ± 42.8 pg/mL in plasma and 129.8 ± 54.2 pg/mL and 139.6 ± 51.5 pg/mL in the CSF. No correlations was found between plasma and CSF IL-1β concentration after LPS in both groups. The QAlb calculated before LPS and 6 h after was similar in all groups. Melatonin did not affected mRNA expression of Il1B, Il1r1 and Il1r2 in the CP. The mRNA expression of Mmp3 and Mmp9 was not detected. Conclusions: The lack of correlation between plasma and CSF IL-1β concentration indicates that at the beginning of inflammation the local synthesis of IL-1β in the CP is an important source of IL-1β in the CSF. Melatonin from slow-release implants does not affect IL-1β concentration in plasma and CSF in early stage of systemic inflammation.