Effect of Caffeine on the Inflammatory-Dependent Changes in the GnRH/LH Secretion in a Female Sheep Model.

Caffeine is one of the most widely consumed psychoactive drugs in the world. It easily crosses the blood-brain barrier, and caffeine-interacting adenosine and ryanodine receptors are distributed in various areas of the brain, including the hypothalamus and pituitary. Caffeine intake may have an impact on reproductive and immune function. Therefore, in the present study performed on the ewe model, we decided to investigate the effect of peripheral administration of caffeine (30 mg/kg) on the secretory activity of the hypothalamic-pituitary unit which regulates the reproductive function in females during both a physiological state and an immune/inflammatory challenge induced by lipopolysaccharide (LPS; 400 ng/kg) injection. It was found that caffeine stimulated (p < 0.01) the biosynthesis of gonadotropin-releasing hormone (GnRH) in the hypothalamus of ewe under both physiological and inflammatory conditions. Caffeine also increased (p < 0.05) luteinizing hormone (LH) secretion in ewes in a physiological state; however, a single administration of caffeine failed to completely release the LH secretion from the inhibitory influence of inflammation. This could result from the decreased expression of GnRHR in the pituitary and it may also be associated with the changes in the concentration of neurotransmitters in the median eminence (ME) where GnRH neuron terminals are located. Caffeine and LPS increased (p < 0.05) dopamine in the ME which may explain the inhibition of GnRH release. Caffeine treatment also increased (p < 0.01) cortisol release, and this stimulatory effect was particularly evident in sheep under immunological stress. Our studies suggest that caffeine affects the secretory activity of the hypothalamic-pituitary unit, although its effect appears to be partially dependent on the animal's immune status.

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.

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.

Stimulatory effect of dopamine derivative, salsolinol, on pulsatile luteinizing hormone secretion in seasonally anestrous sheep: Focus on dopamine, kisspeptin and gonadotropin-releasing hormone.

In the present study, there was testing of the hypothesis that a centrally administered dopamine (DA) derivative, salsolinol, could affect pulsatile luteinizing hormone (LH) secretion in seasonally anestrous sheep by affecting the neuronal components of the estradiol (E2) negative feedback. In two experiments performed during early spring (increasing day length - March/April), salsolinol or Ringer-Locke solution (control) were administered into the third brain ventricle (IIIv): 1) in several injections for three consecutive days; and 2) in several hour-long infusions. In addition to determining the LH concentration (in both experiments), the abundances of gonadotropin-releasing hormone (GnRH) and kisspeptin mRNA were examined in the hypothalamus and LHß subunit mRNA in the pituitary (Experiment 1). In Experiment 2, concentrations of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in perfusates collected from the infundibular nucleus/median eminence (IN/ME) by the push-pull method. In both experiments, salsolinol increased both LH pulse frequency (P < 0.05) and plasma LH concentration (P < 0.001) compared to controls. The injected salsolinol also increased (P < 0.05) the abundance of GnRH mRNA in the mediobasal hypothalamus and kisspeptin mRNA in the arcuate nucleus. The two doses of infused salsolinol decreased DA to undetectable concentrations and DOPAC concentration by 60% in perfusates collected from the IN/ME. In conclusion, exogenous salsolinol functioning centrally stimulates pulsatile LH secretion in sheep during seasonal anestrus. It is suggested that salsolinol may have this effect by reducing the activity of the hypothalamic neuroendocrine dopaminergic system, which results in an increase in both kisspeptin and GnRH neurons activity.

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.

Peripheral injection of SB203580 inhibits the inflammatory-dependent synthesis of proinflammatory cytokines in the hypothalamus.

The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor (TNF) α in the hypothalamus of ewes during prolonged inflammation. Inflammation was induced by the administration of lipopolysaccharide (LPS) (400 ng/kg) over 7 days. SB203580 is a selective ATP-competitive inhibitor of the p38 mitogen-activated protein kinase (MAPK), which is involved in the regulation of proinflammatory cytokines IL-1ß, IL-6 and TNFα synthesis. Intravenous injection of SB203580 successfully inhibited (P < 0.01) synthesis of IL-1ß and reduced (P < 0.01) the production of IL-6 in the hypothalamus. The p38 MAPK inhibitor decreased (P < 0.01) gene expression of TNFα but its effect was not observed at the level of TNFα protein synthesis. SB203580 also reduced (P < 0.01) LPS-stimulated IL-1 receptor type 1 gene expression. The conclusion that inhibition of p38 MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiate new perspectives in the treatment of chronic inflammatory diseases also within the central nervous system. However, potential proinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced very carefully with analysis of all expected and unexpected consequences of treatment.

Effects of central injection of anti-LPS antibody and blockade of TLR4 on GnRH/LH secretion during immunological stress in anestrous ewes.

The present study was designed to examine the effect of intracerebroventricular (icv) administration of antilipopolysaccharide (LPS) antibody and blockade of Toll-like receptor 4 (TLR4) during immune stress induced by intravenous (iv) LPS injection on the gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in anestrous ewes. Injection of anti-LPS antibody and TLR4 blockade significantly (P < 0.01) reduced the LPS dependent lowering amount of GnRH mRNA in the median eminence (ME). Moreover, blockade of TLR4 caused restoration of LH- ß transcription in the anterior pituitary decreased by the immune stress. However, there was no effect of this treatment on reduced LH release. The results of our study showed that the blockade of TLR4 receptor in the hypothalamus is not sufficient to unblock the release of LH suppressed by the immune/inflammatory challenges. This suggests that during inflammation the LH secretion could be inhibited directly at the pituitary level by peripheral factors such as proinflammatory cytokines and circulating endotoxin as well.

The influence of salsolinol on dopaminergic system activity within the mediobasal hypothalamus of anestrous sheep: a model for studies on the salsolinol-dopamine relationship.

Salsolinol with its derivatives has been considered as a potential neurotoxin for the dopaminergic system in the human and rat brain. Investigating a sheep model for studies on the action of salsolinol within the central nervous system we examined whether this compound is able to affect the hypothalamic neuroendocrine dopaminergic (NEDA) system during its high seasonal activity, when sheep entered to anestrus under the long day conditions. Therefore, salsolinol was infused into the third ventricle of the brain in combination with the in vivo push-pull perfusion of the mediobasal hypothalamus/median eminence (MBH/ME). The effects of this drug on either perfusate noradrenaline (NA) or plasma prolactin concentration were also studied. The infusion of salsolinol resulted in rapid and permanent diminution in dopamine (DA) release into the extracellular spaces of the MBH/ME up to an undetectable level and in the 57% decrease in DA metabolite 3,4-dihydroxyphenylacetic acid concentration, compared to the control. This effect of salsolinol was accompanied by the significant enhancement of the pituitary prolactin release into circulation. The concentration of other DA metabolite, homovanillic acid, as well as NA in the MBH/ME was not affected. Thus, our results in the anestrous sheep underline the role played by salsolinol as a neuromodulator for the hypothalamic NEDA system and as a signal transmitter for the pituitary prolactin release. We suggest that the hypothalamic NEDA system of anestrous sheep during its high secretory activity may be set as a model for studies on the salsolinol-dopamine relationship.

Effect of endotoxin on the expression of GnRH and GnRHR genes in the hypothalamus and anterior pituitary gland of anestrous ewes.

An immune/inflammatory challenge can affect reproduction at the level of the hypothalamus, pituitary gland, or gonads. Nonetheless, the major impact is thought to occur within the brain or the pituitary gland. The present study was designed to examine the effect of intravenous (i.v.) lipopolysaccharide (LPS) injection on the expression of gonadotropin-releasing hormone (GnRH) and the gonadotropin-releasing hormone receptor (GnRHR) genes in the hypothalamic structures where GnRH neurons are located as well as in the anterior pituitary gland (AP) of anestrous ewes. We also determined the effect of LPS on luteinizing hormone (LH) release. It was found that i.v. LPS injection significantly decreased GnRH and GnRHR mRNAs levels in the preoptic area (40%, p

Expression of interleukin (IL)-1ß and IL-1 receptors genes in the hypothalamus of anoestrous ewes after lipopolysaccharide treatment.

This study was designed to determine the effect of intravenous lipopolysaccharide (LPS) administration on the secretion of interleukin (IL)-1ß and IL-1 receptors (IL-1Rs) gene expression in the hypothalamus of anoestrous ewes. Gene expression of IL-1ß and its receptors was assayed by the real-time polymerase chain reaction. The expression of IL-1ß in the hypothalamus was detected using Western blot. Our results showed that IL-1ß mRNA is transcribed in the ovine hypothalamus. Lipopolysaccharide increased (p ≤ 0.01) the IL-1ß gene expression in the pre-optic area 2.4-fold, the anterior hypothalamus (AHA) 3.4-fold, the medial basal hypothalamus 3.7-fold and the medial eminence 3.9-fold. The pro-form and mature form of IL-1ß protein were found in the hypothalamus after endotoxin injection. In general, the endotoxin also increased more than two times (p ≤ 0.01) the expression of IL-1 receptor type I (IL-1R1) and type II (IL-1R2) genes in the hypothalamus, except the AHA, where the number of IL-1R2 mRNA was extremely low and not sufficient to the quantitative analysis. These results demonstrate that the peripheral immune/inflammatory challenge increases the IL-1ß expression in the hypothalamus. This endogenous IL-1ß seems to be involved in the modulation of processes which are regulated at the hypothalamic level. One of these processes could be a reproduction.

The role of immunological system in the regulation of gonadoliberin and gonadotropin secretion.

This article reviews data concerning the interactions between immune and neuroendocrine systems in the regulation of reproduction processes at the hypothalamic and pituitary level during immunological stress. Hypothalamus seems to play the most important role in the inhibitory action of immune challenge on the gonadoliberin (GnRH) and gonadotropin secretion as well in the inhibition of the reproductive functions. The administration of endotoxin as a model of immunological stress could alter circulating concentrations of luteinizing hormone (LH) via actions at the hypothalamus through altered GnRH secretion, or at the level of the pituitary via inhibition of LH production and release in response to GnRH. At the central level, interleukin (IL)-1beta seems to play the most important role in the suppression of GnRH secretion during immune challenge. The inhibitory action of this cytokine on GnRH may involve different neural mechanisms: direct action on the GnRH neurons through the IL-1 receptors or indirect action involving other mediators such as opioids, catecholamines, gamma-aminobutyric acid, prostaglandins or nitric oxide.