Acute Effect of Caffeine on the Synthesis of Pro-Inflammatory Cytokines in the Hypothalamus and Choroid Plexus during Endotoxin-Induced Inflammation in a Female Sheep Model.

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-1ß) 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.

Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats.

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.

Effect of Lipopolysaccharide-Induced Inflammatory Challenge on ß-Glucuronidase Activity and the Concentration of Quercetin and Its Metabolites in the Choroid Plexus, Blood Plasma and Cerebrospinal Fluid.

Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.

Chokeberry anthocyanins and their metabolites ability to cross the blood-cerebrospinal fluid barrier.

The aim of this study was to determine whether anthocyanins and their phase II metabolites permeate the blood-cerebrospinal fluid barrier (B-CSF-B) of sheep and to profile these compounds in sheep biofluids after chokeberry intraruminal administration. Anthocyanins were analyzed using micro-HPLC-MS/MS. After chokeberry administration, anthocyanins were absorbed and occurred in body fluids mainly in the form of methylated, glucuronidated, and sulfated derivatives (in total, 21 derivatives were identified). The study showed that anthocyanins penetrated the B-CSF-B and their change in profile and concentration in the cerebrospinal fluid (CSF) resulted from fluctuations in concentrations of these compounds in blood plasma, although the presence of various cyanidin derivatives in CSF also depended on their chemical structure. The biological fate of chokeberry anthocyanins, from absorption into blood to penetration into CSF, was tracked to facilitate the design of further experimental procedures to determine the biological properties of these compounds, including potentially neuroprotective activities.

Photoperiod Affects Leptin Action on the Choroid Plexus in Ewes Challenged with Lipopolysaccharide-Study on the mRNA Level.

The ovine choroid plexus (ChP) expresses the long isoform of the leptin receptor, which makes this structure a potential target for leptin action. In sheep, leptin concentration in plasma is higher during long days (LD) than short days (SD). This study evaluates the influence a of photoperiod on leptin impact on the gene expression of Toll-like receptor 4 (TLR4), proinflammatory cytokines (IL1B, IL6), their receptors (IL1R1, IL1R2, ILRN, IL6R, IL6ST) and inflammasome components necessary for pro-IL-1ß activation (NLRP3, PYCARD, CASP1), chemokine (CCL2), leptin receptor isoforms (LEPRa, LEPRb) and a suppressor of cytokine signalling (SOCS3) in the ChP of ewes treated or not with lipopolysaccharide (LPS). Studies were conducted on adult female sheep divided into four groups (n = 6 in each): control, leptin (20 µg/kg), LPS (400 ng/kg), and LPS and leptin injected under SD and LD photoperiods. The leptin alone did not affect the gene expression but in co-treatment with LPS increased (p < 0.05) IL1B but only during SD, and SOCS3, IL1R2, IL1RN, IL6ST and CCL2 only during LD, and decreased (p < 0.05) the IL1R1 expression only during SD photoperiod. This indicates that the immunomodulatory action of leptin on the ChP is manifested only under the LPS challenge and is photoperiodically dependent.

Effect of Central Injection of Neostigmine on the Bacterial Endotoxin Induced Suppression of GnRH/LH Secretion in Ewes during the Follicular Phase of the Estrous Cycle.

Induced by a bacterial infection, an immune/inflammatory challenge is a potent negative regulator of the reproduction process in females. The reduction of the synthesis of pro-inflammatory cytokine is considered as an effective strategy in the treatment of inflammatory induced neuroendocrine disorders. Therefore, the effect of direct administration of acetylcholinesterase inhibitor-neostigmine-into the third ventricle of the brain on the gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretions under basal and immune stress conditions was evaluated in this study. In the study, 24 adult, 2-years-old Blackhead ewes during the follicular phase of their estrous cycle were used. Immune stress was induced by the intravenous injection of LPS Escherichia coli in a dose of 400 ng/kg. Animals received an intracerebroventricular injection of neostigmine (1 mg/animal) 0.5 h before LPS/saline treatment. It was shown that central administration of neostigmine might prevent the inflammatory-dependent decrease of GnRH/LH secretion in ewes and it had a stimulatory effect on LH release. This central action of neostigmine is connected with its inhibitory action on local pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)α synthesis in the hypothalamus, which indicates the importance of this mediator in the inhibition of GnRH secretion during acute inflammation.

Neostigmine Attenuates Proinflammatory Cytokine Expression in Preoptic Area but Not Choroid Plexus during Lipopolysaccharide-Induced Systemic Inflammation.

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-) 1ß, 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.

Plasma and cerebrospinal fluid interleukin-1ß during lipopolysaccharide-induced systemic inflammation in ewes implanted or not with slow-release melatonin.

BACKGROUND: Interleukin-1ß (IL-1ß) 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-1ß in ewes and its correlation with plasma during inflammation as well as melatonin action on the concentration of IL-1ß 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-1ß and albumin concentration were measured. To assess the functions of brain barriers albumin quotient (QAlb) was used. Expression of IL-1ß (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-1ß 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-1ß 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-1ß concentration indicates that at the beginning of inflammation the local synthesis of IL-1ß in the CP is an important source of IL-1ß in the CSF. Melatonin from slow-release implants does not affect IL-1ß concentration in plasma and CSF in early stage of systemic inflammation.

Peripheral Inhibitor of AChE, Neostigmine, Prevents the Inflammatory Dependent Suppression of GnRH/LH Secretion during the Follicular Phase of the Estrous Cycle.

The study was designed to test the hypothesis that the inhibition of acetylcholinesterase (AChE) activity at the periphery by Neostigmine (0.5 mg/animal) will be sufficient to prevent inflammatory dependent suppression of the gonadotropin-releasing hormone (GnRH)/luteinising hormone (LH) secretion in ewes in the follicular phase of the estrous cycle, and this effect will be comparable with the systemic AChE inhibitor, Donepezil (2.5 mg/animal). An immune/inflammatory challenge was induced by peripheral administration of lipopolysaccharide (LPS; 400 ng/kg). Peripheral treatment with Donepezil and Neostigmine prevented the LPS-induced decrease (P < 0.05) in LHß gene expression in the anterior pituitary gland (AP) and in LH release. Moreover, Donepezil completely abolished (P < 0.05) the suppressory effect of inflammation on GnRH synthesis in the preoptic area, when pretreatment with Neostigmine reduced (P < 0.05) the decrease in GnRH content in this hypothalamic structure. Moreover, administration of both AChE inhibitors diminished (P < 0.05) the inhibitory effect of LPS treatment on the expression of GnRH receptor in the AP. Our study shows that inflammatory dependent changes in the GnRH/LH secretion may be eliminated or reduced by AChE inhibitors suppressing inflammatory reaction only at the periphery such as Neostigmine, without the need for interfering in the central nervous system.

PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) differentially affects the VEGF/VEGFR system depending on photoperiod in the ovine choroid plexus.

Ortho-substituted polychlorinated biphenyls (PCBs) preferentially accumulate in the brain and cerebrospinal fluid (CSF) compared with other PCBs. We previously demonstrated in ewes that an identical dose of PCB153, the most environmentally prevalent congener, resulted in a higher plasma concentration during short days (SD: 1200pg/ml) than during long days (LD: 200pg/ml). Moreover, PCB153 treatment only reduced the SD tight junction protein content in the choroid plexus (CP), which was followed by a significant increase of the PCB153 concentration in the CSF. The aim of the present study was to evaluate how PCB153 treatment affects the VEGF/VEGFR system that maintains CSF homoeostasis and CP function. To do so, we collected CPs from ovariectomised, oestradiol-replaced adult ewes maintained under artificial LD or SD and treated them per os with low doses of PCB153 (0.3mg/kg, 3 times a week for 3 weeks). Exposure to PCB153 significantly affected (P<0.05) the VEGF/VEGFR system during the SD period, provoking increases in VEGF164 mRNA and protein levels and decreases in VEGFR-1 mRNA levels and VEGFR-2 mRNA and protein levels. These results suggest that exposure to environmentally relevant dose of PCB153 affects the VEGF/VEGFR system, which is involved in the fenestration of the CP endothelium and therefore in CSF production.

Quercetin and isorhamnetin aglycones are the main metabolites of dietary quercetin in cerebrospinal fluid.

SCOPE: Reports on the protective effect of certain foods on brain functions are numerous; however, the permeability of the brain barriers by food components is still hardly recognised. There have been in vitro studies aimed at demonstrating this possibility, but not much is known about this phenomenon in in vivo systems. The objective of the study was to determine the metabolites of dietary quercetin (Q) in urine, blood plasma and cerebrospinal fluid (CSF) after intra-rumen administration of Q rich onion dry skin in an animal model. METHODS AND RESULTS: Eleven sheep had permanently implanted cannulas in the third ventricle of the brain as the means for CSF collection. The animals were administered Q at the dose of 10 mg/kg bwt. For 12 h the concentration of Q metabolites was measured in urine, blood plasma, and CSF. It was demonstrated that while in blood plasma Q and isorhamnetin mono-glucuronides or mono-sulphates were the main metabolites (80%), in CSF their aglycones were the dominating ones (88%). CONCLUSION: Q and IR aglycones are the main Q metabolites present in CSF after dietary Q intake. Their passive transport through blood-CSF barrier or a de-conjugating mechanism within that barrier may be involved.

Caffeine stimulates in vitro pituitary LH secretion in lipopolysaccharide-treated ewes.

The study was designed to determine the effects of caffeine on luteinizing hormone (LH) secretion and gene expression of caffeine-associated receptors in anterior pituitary (AP) explants obtained from saline- and lipopolysaccharide (LPS)-treated ewes. Animals had been treated with LPS or saline daily for seven days. Three hours after the last injection of LPS/saline, the AP were collected and divided into four explants. The explants were incubated with: 1/medium-199 (control explants), 2/gonadotropin-releasing hormone (GnRH; 100 pmol/mL; a positive control), 3/caffeine (10 mmol/L), or 4/GnRH+caffeine. Caffeine stimulated (p<0.05) LH release by explants from both saline (19.7 vs. control 12.6 ng/mg) and LPS (28.3 vs. control 13.9 ng/mg) treated animals. The effect of caffeine on LH secretion was stronger in the LPS-treated group than in saline-treated group, and the observed LH release was similar to that induced by GnRH alone (27.2 ng/mg). Caffeine increased (p<0.05) LHß gene expression only in explants from LPS-treated animals. In conclusion, the results of the present study demonstrated a stimulatory in vitro effect of caffeine on LH secretion by ovine pituitary explants. The potency of the caffeine-induced LH secretion was affected by in vivo treatment of the animals with endotoxin.

Profile of toll-like receptor mRNA expression in the choroid plexus in adult 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.

Effect of a two-week treatment with a low dose of 2,2'4,4',5,5'-hexachlorobiphenyl (PCB153) on tight junction protein expression in ovine choroid plexus during long and short photoperiods.

Ortho-substituted polychlorinated biphenyls (PCBs) preferentially accumulate in the brain and cerebrospinal fluid (CSF) compared to other PCBs. We previously reported that the same dose of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) induced higher concentrations in the CSF of treated animals compared to controls during short days (SD), while no differences were observed during long days (LD). Similarly, the plasma concentration of PCB153 in treated ewes was higher during SD than LD. To understand the structural and molecular events explaining the photoperiodically different concentration of PCBs in the CSF in sheep, we studied the effect of photoperiod on PCB153 action on tight junction (TJ) protein expression in the choroid plexus (CP) of ewes. For that purpose, we collected CP from ovariectomised, estradiol-treated ewes maintained under artificial LD or SD and orally administered with a low dose (0.33 mg/kg/day, 3 times per week for 3 weeks) of PCB153 or vehicle. Exposure to PCB153 affected TJ proteins only during SD, and the levels of claudin-1, zonula occludens-2 (ZO-2), and afadin (AF-6) were significantly lower when compared to vehicle-treated animals. No differences were observed for occludin, junctional adhesion molecule-1 (JAM-1), claudin-5, ZO-1 and ZO-3. There was no effect of PCB153 treatment on TJ-mRNA levels. These results indicate that PCB153 selectively alters TJ proteins in the ovine CP. These alterations appear to be associated with the level of PCB153 in the blood plasma, which is modulated by the photoperiod. This study emphasises the importance of photoperiod in the susceptibility of adult sheep to PCBs.

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods.

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.

Photoperiod modulates access of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) to the brain and its effect on gonadotropin and thyroid hormones in adult ewes.

The effects of photoperiod on the cerebrospinal fluid (CSF) concentration of six ortho-substituted polychlorinated biphenyls (PCBs: PCB28, PCB52, PCB101, PCB138, PCB153, and PCB180), the effects of an orally administered low dose of PCB153 (0.3mg/kg, three times a week for three weeks) on PCBs and thyroid hormones (THs) concentrations in the CSF and plasma, and the release of luteinizing hormone (LH) were determined in ovariectomized, estradiol-implanted ewes (2.5 years old) maintained indoors under artificial long day (LD, 16L: 8D) and short day (SD, 8L: 16D) conditions. Concentrations of two PCBs (PCB28 and PCB153) in the plasma and four PCBs in the CSF (PCB101, PCB138, PCB153, and PCB180) were significantly higher during LD than SD. Following PCB153 treatment, its concentration in the plasma was higher in SD (1.2 ± 0.3 ng/ml) than LD (0.2 ± 0.05 ng/ml), but similar in the CSF (10.2 ± 3.7 pg/ml vs. 13 ± 0.7 pg/ml) under both photoperiods. During SD, the concentration of PCB153 in the CSF was higher in treated animals than controls, while no differences were noted under LD. These findings indicate that in ewes, exposure of the brain to more highly chlorinated, ortho-substituted PCBs may be modulated by photoperiod. PCB153 treatment had no effect on plasma THs, but reduced total triiodothyronine concentration during LD and free thyroxine during SD in the CSF. Under both photoperiods, PCB153 reduced basal plasma LH and reinforced the inhibition of pulsatile LH release during LD. As PCB153 reduced LH and THs (which are involved in the seasonal control of reproduction in ewes), it may have a braking effect on seasonal transitions between active and inactive phases of reproduction.

Tight junction proteins vary in the choroid plexus of ewes according to photoperiod.

Sheep from temperate latitudes exhibit seasonal variations in many physiological functions such as reproduction, food intake, body weight, and pelage growth. Majority of seasonal changes are controlled by the annual photoperiodic cycle and melatonin secretion. For reproduction, the resulting key event is a modulation of the negative feedback of steroids on gonadotropin secretion. However, this seasonal effect could also depend on variable uptake of steroids by the brain. Seasonal regulation of food intake also involves numerous peripheral hormones, among which the protein hormone leptin informs the brain on the metabolic status of the animal. It has been shown previously that access of progesterone, estradiol and leptin to the cerebrospinal fluid (CSF) increases under long days. This physiological modulation of the passage of hormones to the brain could depend on regulation of the permeability of the blood-CSF barrier. This study therefore compared the tight junction proteins in the choroid plexus of ewes exposed to short days or long days. Levels of occludin, zonula occludens proteins (ZO) ZO-1 and ZO-2, afadin and cadherin were significantly higher during short days, but no statistical difference was observed for junctional adhesion molecule 1 (JAM-1), ZO-3 or claudins 1 and 5. These results are consistent with an increase in the blood-CSF barrier permeability during long days through a regulation of tight junctions and show that the permeability could depend upon physiological conditions such as photoperiodic status.

The choroid plexus--cerebrospinal fluid system: undervaluated pathway of neuroendocrine signaling into the brain.

The cerebrospinal fluid (CSF) is a major part of the extracellular fluid of the central nervous system. The function of the CSF and the tissue that secretes it, the choroid plexus (CP) has traditionally been thought as providing the brain with essential nutrients, removing products of neuronal activity of the central nervous system, and providing mechanical support for the brain's fragile cellular network. More recent studies suggest, however, that the CP and CSF system play a much more active role in the function of the central nervous system being a target, source and pathway for neuroendocrine signaling within the brain.

Luteinising hormone attenuates the vascular response to norepinephrine.

The present study examines the direct effect of luteinising hormone (LH) on the reactivity of the porcine uterine artery to norepinephrine (NE). Three-mm-long arterial segments collected during the luteal phase of the oestrous cycle were mounted in an organ bath for isometric tension recording. After 30 min of equilibration in optimal passive tone, one part of the vessels was treated with 10 ng/ml of LH in PBS (experimental), while a second part of the arterial segments was treated with 10 ng/ml of bovine serum albumin (BSA) in PBS (control). After 30 min of equilibration, NE was given to each organ bath in a cumulative concentration manner, ranging between 1 x 10(-8) mol/l to 3 x 10(-4) mol/l. NE caused a dose-dependent contraction of all experimental and control arteries. The addition of LH caused a rightward shift of the dose-response curve to NE. The corresponding EC50 values were 2.17 (+/- 0.39) micromol/l in PBS-pretreated vessels and 3.35 (+/- 0.41) micromol/l in LH-pretreated vessels (P < 0.05). The results of the present study demonstrate that LH attenuates the vascular response to NE in third-order branches of the uterine artery. Therefore, it can be suggested that besides the known effect of LH-hCG on the formation of vasoactive eicosanoids, an additional mechanism is involved in the direct action of LH on blood flow in the uterine arteries in pigs.

Luteinizing hormone and prolactin are not retrograde transferred in perihypophyseal vascular complex in ewes.

The objective of the study was to determine whether luteinizing hormone (LH) and prolactin (PRL) can access the brain by way of transfer from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. Studies were performed on heads of 22 mature sheep isolated during different phases of the estrous cycle and perfused with autologous blood. We were not able to demonstrate any transfer of LH and PRL in the investigated periods. This suggests that molecular weight of hormone may be a main factor determining the permeation and transfer of hormones in the perihypophyseal vascular complex.