SQUID-based detection of ultra-low-field multinuclear NMR of substances hyperpolarized using signal amplification by reversible exchange.

Ultra-low-field (ULF) nuclear magnetic resonance (NMR) is a promising spectroscopy method allowing for, e.g., the simultaneous detection of multiple nuclei. To overcome the low signal-to-noise ratio that usually hampers a wider application, we present here an alternative approach to ULF NMR, which makes use of the hyperpolarizing technique signal amplification by reversible exchange (SABRE). In contrast to standard parahydrogen hyperpolarization, SABRE can continuously hyperpolarize 1 H as well as other MR-active nuclei. For simultaneous measurements of 1 H and 19 F under SABRE conditions a superconducting quantum interference device (SQUID)-based NMR detection unit was adapted. We successfully hyperpolarized fluorinated pyridine derivatives with an up to 2000-fold signal enhancement in 19 F. The detected signals may be explained by two alternative reaction mechanisms. SABRE combined with simultaneous SQUID-based broadband multinuclear detection may enable the quantitative analysis of multinuclear processes.

Asthma is associated with reduced fibrinolytic activity, abnormal clot architecture, and decreased clot retraction rate.

The aim of this study was to assess whether steroid-naïve asthma modulates hemostasis. We evaluated the clot retraction rate (CRR), fibrinolysis rate (FR), clot density (CD) (by confocal microscopy), plasma levels of plasminogen activator inhibitor (PAI-1), and factor XIII (FXIII), NO in exhaled breath (FENO ), spirometry (FEV1 ) and eosinophil count (EOS) in 36 patients with allergic, steroid-naïve asthma and in 34 healthy controls. We observed significantly (P < 0.001) reduced CRR, FR, and FEV1 and increased FENO , EOS, PAI-1, FXIII, and CD in patients with asthma compared with controls. In patients with asthma, FR negatively correlated with CD, FXIII, PAI-1, FENO , and EOS and positively with FEV1 . FXIII positively correlated with CD. Clot retraction rate negatively correlated with FENO and positively with FEV1 (all P < 0.001). These novel findings suggest that asthma itself is associated with decreased CRR and reduced fibrinolytic potential resulting from alterations in clot architecture and elevated levels of plasma FXIII and PAI-1.

Salsolinol-a potential inhibitor of the gonadotropic axis in sheep during lactation.

This study tested the hypothesis that salsolinol, a derivative of dopamine, affects GnRH and LH secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus-median eminence of sheep at the fifth wk of lactation to antagonize salsolinol's action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing 2 doses of salsolinol, 20 and 100 µg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHß subunit messenger RNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (P < 0.001 and P < 0.05, respectively) compared with those in controls. Significantly higher LH concentrations occurred during the first (P < 0.001), second (P < 0.001), and fourth (P < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared with that in the control (P < 0.01 and P < 0.001, respectively). Salsolinol had no effect on the relative LHß subunit messenger RNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses does not allow to exclude the central action of salsolinol.

Involvement of salsolinol in the suckling-induced oxytocin surge in sheep.

During lactation, the main surge of oxytocin is induced by a suckling stimulus. Previous studies have shown that salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), a dopamine-derived compound, stimulates both the synthesis and the release of oxytocin in lactating sheep. The objective of the present study was to verify the hypothesis that salsolinol is involved in the mechanism that generates the oxytocin surge that occurs during suckling. Thus, a structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), known to antagonize some of its actions, was infused into the third ventricle of the brain of lactating sheep nursing their offspring. Serial 30-min infusion of 1MeDIQ (4 × 60 µg/60 µL) or vehicle were administered at 30-min interval from 10 AM to 2 PM. The experimental period in every ewe consisted of a nonsuckling period (10 AM-12 PM) and a suckling period (12 PM-2 PM). Blood samples were collected every 10 min, to measure plasma oxytocin concentration by RIA. In control sheep, oxytocin surges of high amplitude were observed during the suckling period. The oxytocin surges induced by suckling were significantly (P < 0.01) diminished in sheep receiving 1MeDIQ infusions as compared to those that received control infusions. However, no significant effect of 1MeDIQ was observed on basal oxytocin release, before suckling. Furthermore, oxytocin release, as measured by the area under the hormone response curve (AUC), was significantly decreased by the administration of 1MeDIQ during the suckling period. This study shows that elimination of the effect of salsolinol within the central nervous system of lactating sheep attenuates the oxytocin surge induced by suckling. Therefore, salsolinol may be an important factor in the oxytocin-stimulating pathway in lactating mammals.

Salsolinol Up-Regulates Oxytocin Expression and Release During Lactation in Sheep.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a dopamine-derived compound present in the central nervous system and pituitary gland. Several previous studies on lactating sheep and rats have reported that salsolinol plays a crucial role in the regulation of prolactin secretion. The present study investigated the effects of salsolinol, which was infused into the third ventricle of the brain, on oxytocin expression and release in lactating sheep, 48 h after weaning of 8-week-old lambs. Serial 30-min infusions of salsolinol and vehicle were performed at 30-min intervals from 10.00 to 15.00 h. Blood samples were collected every 10 min. The supraoptic nucleus (SON), paraventricular nucleus (PVN) and posterior pituitary were collected immediately after the experiment. Expression levels of mRNAs for oxytocin and peptidylglycine α-amidating monooxygenase (PAM), the terminal enzyme in the oxytocin synthesis pathway, were measured using a real-time polymerase chain reaction. Oxytocin peptide content in the posterior pituitary was measured by an enzyme-linked immunosorbent assay, and plasma oxytocin concentration was measured by radioimmunoassay. Salsolinol treatment significantly up-regulated oxytocin and PAM gene expression in the SON (P < 0.01 and P < 0.05, respectively), PVN (P < 0.01 and P < 0.05, respectively) and posterior pituitary (P < 0.05 and P < 0.05, respectively). Oxytocin peptide content in the posterior pituitary and the area under the response curve of plasma oxytocin were significantly (P < 0.05 and P < 0.01, respectively) higher in salsolinol-treated sheep than in control animals. The present study shows for the first time that salsolinol stimulates oxytocin secretion during lactation in sheep.

Salsolinol: a potential modulator of the activity of the hypothalamic-pituitary-adrenal axis in nursing and postweaning sheep.

The most well-known physiological action of salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is the stimulation of prolactin secretion, especially during lactation. In addition, our recent work demonstrated that salsolinol inhibits the stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis in lactating sheep. Here, we investigated whether salsolinol regulates the basal activity of the HPA axis in lactating sheep and whether its inhibitory action on the stress-induced activity of the HPA axis is present during the postweaning period. The first experiment was performed during the fifth week of lactation, in which unstressed sheep received an intracerebroventricular infusion of an antagonistic analogue of salsolinol, 1-MeDIQ (1-methyl-3,4-dihydroisoquinoline). Simultaneously, the infundibular nucleus and/or median eminence was perfused using the push-pull method. Sheep that received 1-MeDIQ infusion showed significantly higher concentration of plasma ACTH during the second, third, and fourth hour (P < 0.001, P < 0.01, and P < 0.001, respectively) and cortisol during the third and fourth hour (P < 0.001 and P < 0.01, respectively) than did sheep that received control infusion. There was no significant difference in the mean perfusate corticotropin-releasing hormone concentration between the 1-MeDIQ and control treatments. In the second experiment, sheep received an intracerebroventricular infusion of salsolinol during the ninth week of lactation and 48 h after lamb weaning. A comparison between the control groups in the first and second experiments revealed that sheep after weaning (ninth week of lactation) had significantly higher mean ACTH (P < 0.001) and cortisol (P < 0.001) concentrations during the first 2 h of the experiment than the nursing females (fifth week of lactation) had. Salsolinol significantly reduced the increased concentrations of ACTH and cortisol (P < 0.01) in sheep after lamb weaning. However, there was no difference in the expression of proopiomelanocortin messenger RNA within the anterior pituitary between the control and salsolinol-treated groups. In conclusion, salsolinol regulates the basal activity of the HPA axis in lactating sheep. In addition, the HPA axis of postweaning females is more sensitive to stressors associated with the experimental procedures, and salsolinol attenuates ACTH and cortisol release in this phenomenon.

Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria.

Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 µM), platelet-rich plasma (PRP) threshold concentration (t.c. 50 µM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25-100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and α angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target.

Suckling and salsolinol attenuate responsiveness of the hypothalamic-pituitary-adrenal axis to stress: focus on catecholamines, corticotrophin-releasing hormone, adrenocorticotrophic hormone, cortisol and prolactin secretion in lactating sheep.

In mammals, the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to stress is reduced during lactation and this mainly results from suckling by the offspring. The suckling stimulus causes a release of the hypothalamic 1-metyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) (a derivative of dopamine), one of the prolactin-releasing factors. To investigate the involvement of salsolinol in the mechanism suppressing stress-induced HPA axis activity, we conducted a series of experiments on lactating sheep, in which they were treated with two kinds of isolation stress (isolation from the flock with lamb present or absent), combined with suckling and/or i.c.v infusion of salsolinol and 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ; an antagonistic analogue of salsolinol). Additionally, a push-pull perfusion of the infundibular nucleus/median eminence (IN/ME) and blood sample collection with 10-min intervals were performed during the experiments. Concentrations of perfusate corticotrophin-releasing hormone (CRH) and catecholamines (noradrenaline, dopamine and salsolinol), as well as concentrations of plasma adenocorticotrophic hormone (ACTH), cortisol and prolactin, were assayed. A significant increase in perfusate noradrenaline, plasma ACTH and cortisol occurred in response to both kinds of isolation stress. Suckling and salsolinol reduced the stress-induced increase in plasma ACTH and cortisol concentrations. Salsolinol also significantly reduced the stress-induced noradrenaline and dopamine release within the IN/ME. Treatment with 1-MeDIQ under the stress conditions significantly diminished the salsolinol concentration and increased CRH and cortisol concentrations. Stress and salsolinol did not increase the plasma prolactin concentration, in contrast to the suckling stimulus. In conclusion, salsolinol released in nursing sheep may have a suppressing effect on stress-induced HPA axis activity and peripheral prolactin does not appear to participate in its action.

Nitric oxide scavenging by cell-free hemoglobin may be a primary factor determining hypertension in polycythemic patients.

We tested the hypothesis that hypertension associated with polycythemia vera (PV) may be related to hemoglobin released from erythrocytes (cell-free hemoglobin, fHb). We assessed hematocrit, mean arterial pressure (MAP), blood viscosity, and the level of fHb and nitrite/nitrate (NOx) in the plasma of 73 PV patients and 38 healthy controls. The effect of isovolemic erythrocytapheresis (ECP) on the considered parameters was also studied. From the whole group of PV patients a subset of subjects with normal (normotensive patients, n = 16) and elevated MAP (hypertensive patients, n = 57) can be subtracted. It was found that in comparison with healthy controls, PV patients have significantly (p ≤ 0.01) elevated Hct (0.567 vs. 0.422), blood viscosity (5.45 vs. 3.56 cP), MAP (106.8 vs. 93.8 mmHg), plasma fHb (9.7 vs. 2.8 mg/dL), and NOx levels (34.1 vs. 27.5 µM). Compared with normotensive patients, hypertensive PV patients demonstrated a higher rise in fHb (10.2 vs. 8.0) and plasma NOx levels (35.8 vs. 31.0). In PV patients, fHb positively correlates with MAP (r = 0.489), NOx levels (r = 0.461), hematocrit (r = 0.428), and viscosity (r = 0.393). Blood viscosity positively correlated with hematocrit (r = 0.894), but not with other considered parameters. In PV patients MAP poorly correlated with hematocrit, whereas the correlation between MAP and NOx altered from - 0.325 (healthy control) to + 0.268 (PV patients). ECP procedure was associated with a significant (p < 0.01) reduction of hematocrit, fHb, blood viscosity, and MAP. In the normotensive subgroup of PV patients the ECP procedure did not affect MAP. It can be concluded that accelerated scavenging of nitric oxide by fHb rather than high Hct may be a key factor determining the development of hypertension in PV patients.

Different types of opioid receptors involved in the suppression of LH secretion in lactating sheep.

To learn the involvement of endogenous opioid peptides (EOP) in the regulation of reproductive activity in ruminants, the effects of different opioid antagonists on luteinizing hormone (LH) secretion were determined in sheep during the early stage of lactation. The opioid receptor antagonists: naloxone (all types of receptors, n=5), naloxonazine (µ receptor, n=5), GNTI- (κ receptor, n=5), naltrindole (δ receptor, n=5) or the vehicle (control, n=5) were infused intracerebroventricularly in a series of five 30-min infusions (60µg/60µl) at 30-min intervals. The period of the experiment included the non-suckling (10:00-12.30) and suckling (12.30-15.00) periods. Blood samples were collected from 10.00 to 15.00 at 10-min intervals, and plasma LH concentration was assayed by the radioimmunoassay method. The obtained results showed that blocking of the EOP action within the central nervous system in lactating sheep caused a significant (p<0.001) increase in LH concentration in all treated groups, in comparison to the control. In the naloxone-treated group, a significant (p<0.05) increase in LH secretion also occurred during suckling. The amplitude of LH pulses increased significantly in the naloxonazine- (p<0.01) and naltrindole- (p<0.05) treated ewes compared to the control; there were no significant differences in the frequency of LH pulses among the groups. In conclusion, our study indicates that EOP play a crucial role in the mechanism inhibiting GnRH/LH axis activity in lactating sheep and that the ligands for µ opioid receptor may have the highest inhibitory effect.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes.

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from -2 to -0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1µmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHß and GnRH-R in the pituitary. In the case of the GnRH and LHß genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

Seasonal changes in the interactions among leptin, ghrelin, and orexin in sheep.

The adaptation of the physiology of an animal to changing conditions of light and food availability is evident at the behavioral and hormonal levels. Melatonin, leptin, ghrelin, and orexin, which exhibit rhythmic secretion profiles under ad libitum feeding conditions, are sensitive to changes in daylength, forming a tight web of interrelationships in the regulation of energy balance. The aim of this study was to determine the effects of central injections of leptin, ghrelin, and orexin on the reciprocal interactions among these hormones and the influence of photoperiod on these responses. Twenty-four ovariectomized and estradiol-implanted ewes were used in a replicated switchback design. The ewes were assigned randomly to 1 of 6 treatment groups, and the treatments were infused into their third ventricles 3 times at 0, 1, and 2 h, with 0 h being at dusk. The treatments were as follows: 1) control, Ringer-Locke buffer; 2) leptin, 0.5 µg/kg BW; 3) ghrelin, 2.5 µg/kg BW; 4) orexin B, 0.3 µg/kg BW; 5) leptin antagonist, 50 µg/kg BW, then ghrelin, 2.5 µg/kg BW; and 6) leptin antagonist, 50 µg/kg BW, then orexin B, 0.3 µg/kg BW. Blood samples (5 mL) were collected at 15-min intervals for 6 h. The administration of leptin increased (P < 0.05) plasma concentrations of melatonin during short-day (ShD) photoperiods and decreased (P < 0.05) them during long-day (LD) photoperiods, whereas ghrelin decreased (P < 0.05) melatonin concentrations during ShD photoperiod, and orexin had no effect (P > 0.1). Leptin attenuated (P < 0.05) ghrelin concentrations relative to the concentration in controls during ShD. The plasma concentrations of orexin were reduced (P < 0.05) after leptin infusions during LD and ShD photoperiods; however, ghrelin had the opposite effect (P < 0.05) on orexin concentration. Orexin increased (P < 0.05) ghrelin concentrations during LD. Ghrelin and orexin concentrations were increased (P < 0.05) after leptin antagonist infusions. Our data provide evidence that the secretion of leptin, ghrelin, and orexin are seasonally dependent, with relationships that are subject to photoperiodic regulation, and that leptin is an important factor that regulates ghrelin and orexin releases in sheep.

Regulation of growth hormone secretion in nursing ewes: an involvement of µ-receptor subtype.

Suckling by newborns induces a surge of lactogenic hormones, that is prolactin and growth hormone (GH), in mother's body, with endogenous opioid peptide (EOP) participating in generation of this surge. The aim of the current study was to investigate which types of opioid receptors are involved in generation of the GH surge in ewes during suckling. A series of intracerebroventricular infusions of opioid receptors antagonists: naloxone (for all types of receptors), naloxonazine (specific for µ receptor) and 5'-guanidinonaltrindole (GNTI--specific for κ receptor) and the vehicle (control) were performed in nursing sheep during the fifth week of lactation. All infusions were carried out in a serial manner: five 30-min infusions (60 µg/60 µl) from 10:00 to 15:00, at 30-min intervals. The period of the experiment consisted of the non-suckling (10:00-12:30) and suckling (12:30-15:00) periods. Simultaneously, blood samples were collected at 10-min intervals to determine plasma GH concentration by radioimmunoassay. Suckling evoked a rapid increase in GH concentration in control ewes. Naloxone and naloxonazine significantly decreased both the basal GH release in the non-suckling period and the suckling-induced GH surge. Specifically, the suppressive effect concerned either the duration or the amplitude of the GH surge. In contrast, GNTI did not significantly affect the GH release. In conclusion, the EOPs may affect the regulatory process of GH secretion in lactating sheep, especially through µ opioid receptor.

Central injection of exogenous IL-1ß in the control activities of hypothalamic-pituitary-gonadal axis in anestrous ewes.

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1ß on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, ß subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1ß on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1ß (10 or 50 µg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 µg (60%; p ≤ 0.01) but not with the 10 µg dose was observed. The centrally administrated IL-1ß lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1ß injection upon the release of LH and FSH. However, the central injection of IL-1ß strongly decreased the LHß mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHß mRNA in the case of the 50 µg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1ß is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.

Seasonal effects of central leptin infusion and prolactin treatment on pituitary SOCS-3 gene expression in ewes.

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 µg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

Opioid-salsolinol relationship in the control of prolactin release during lactation.

Endogenous opioid peptides (EOP) and dopamine (DA)-derived salsolinol are implicated in the suckling-induced prolactin surge. The aim of this study was to investigate the relationship between the opioidergic and salsolinergic activity in the mediobasal hypothalamus of nursing sheep. The sheep were infused intracerebroventricularly with opioid receptors antagonists: naloxone (all types of receptors, n=6); naloxonazine (µ receptor, n=6) or the vehicle (control, n=6) in a series of five 30-min infusions (60 µg/60 µl) from 10:00 to 15:00, at 30-min intervals. The period of the experiment included the non-suckling (10:00-12:30) and suckling (12:30-15:00) periods. Simultaneously, a push-pull perfusion of the infundibular nucleus/median eminence was performed in every sheep to study the dopaminergic system activity. Blood samples were also collected at 10-minute intervals to determine plasma prolactin concentration. Both the mean perfusate salsolinol and plasma prolactin concentrations were higher during the suckling vs. non-suckling (P<0.001) period in the control. The perfusate DA concentration was below the detection limit in this group. Treatment with either naloxone or naloxonazine significantly (P<0.01) diminished plasma prolactin concentration, as compared with the controls and blocked the prolactin surge during suckling. In drug-infused sheep, the perfusate salsolinol concentration was below the detection limit but the increased DA and its metabolite 3,4-dihydroxyphenylacetic acid concentrations were observed. In conclusion, the stimulatory action of EOP on prolactin secretion in nursing females is mediated, at least in part, by salsolinol, and the ligands for µ opioid receptor may be the primary factors of this relationship, especially with respect to the suckling-induced prolactin surge.

Independent changes of thyroid hormones in blood plasma and cerebrospinal fluid after melatonin treatment in ewes.

The authors measured the effects of exogenous melatonin treatment on the concentrations of total (T) and free (f) fractions of thyroxine (T4) and triiodothyronine (T3) in cerebrospinal fluid (CSF) and blood plasma as well as the expression of their binding/transporter protein, transthyretin (TTR), in the choroid plexus of ewes from May to August. Melatonin implantation in May and July mainly prevented the decrease in plasma for fT3 and TT3 exhibited in untreated group, and induced a limited decrease in TT4 in June. By contrast, melatonin implantation prevented the decrease in CSF fT3 observed in the untreated group. No effect of melatonin was found on the expression of TTR mRNA in the choroid plexus There were a correlations between blood fT4 and CSF TT4 concentrations in both control and melatonin treated group (r(2)-0.4; P < 0.01 vs. r(2)-0.14; P < 0.05), as well as between blood fT3 and CSF TT3 concentrations but only in the melatonin-treated group (r(2)-0.26; P < 0.02). We conclude that T3, the active form of the hormone within the brain, is regulated by melatonin independently of the peripheral changes within the blood. The lack of correlation between plasma fT3 and CSF TT3 in the control group suggests that an increase in local T3 conversion could contribute as an additional source of T3 in the CSF during the period of increasing day length. These data seem to confirm a local nature for recently discovered connections between the pineal melatonin signal and thyroid-dependent seasonal biology in mammals.

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.

Thyroid hormones in the cerebrospinal fluid of the third ventricle of adult female sheep during different periods of reproductive activity.

Thyroid hormones (THs) are obligatory for transition from breeding season to anestrus in sheep. In this process, THs act during a very limited time of the year and primarily within the brain. In ewes chronically equipped for sampling cerebrospinal fluid (CSF) from the third ventricle, we have characterized the concentrations of total and free thyroxine (T4), triiodothyronine (T3), and total reverse T3 (rT3) in the CSF during breeding season, anestrus and during a critical period required for transition to anestrus (December-March). The total T4, T3, rT3 and free T3 average concentrations (+/- SEM) in CSF were 1.5 +/- 0.07 ng/ml, 14.5 +/- 1.2 pg/ml, 43 +/- 7.4 pg/ml, and 0.6 +/- 0.05 pg/ml, respectively, and all were significantly lower (p < 0.001) than in blood plasma except free T4 (12.6 +/- 1.1 pg/ml), which was similar to that in plasma. There was a seasonal trend (p < 0.05) in the concentration of total T3 (highest in December) and free T4 (highest in November) in the CSF that does not follow that in blood plasma. During the period of transition to anestrus the CSF total T3/TT4 molar ratio and free T3/T4 ratio were significantly lower (p < 0.05 and p < 0.01, respectively) than in blood plasma, while the total rT3/T4 ratio was significantly higher (p < 0.01) at the end of this period (March). Additionally, the CSF total rT3 concentrations were also significantly correlated with the CSF total T4 levels (r = 0.57; p < 0.05). In conclusion, the CSF in sheep may serve as a considerable source of thyroid hormones for neuroendocrine events. The lack of significant changes in THs concentrations in the CSF during the period of transition to anestrus indicate that neither seasonal changes of THs circulating in the blood plasma nor THs circulating in the CSF actively drive the transition to anestrus.

The possible involvement of salsolinol and hypothalamic prolactin in the central regulatory processes in ewes during lactation.

Salsolinol, a dopamine-related compound and prolactin-producing cells were found in the ovine hypothalamus. This study was designed to test the hypothesis that salsolinol, acting from the CNS level, is able to stimulate pituitary prolactin release as well as prolactin mRNA expression in the anterior pituitary cells (AP) and in the mediobasal hypothalamus (MBH) in lactating ewes. The intracerebroventricular infusions of salsolinol in two doses, total of 50 ng or 5 µg, were performed in a series of five 10-min infusions at 20-min intervals. All infusions were made from 12:30 to 15:00 and the pre-infusion period was from 10:00 to 12.30 h. The prolactin concentration in plasma samples, collected every 10 min, was determined by radioimmunoassay; prolactin mRNA expression in AP and MBH tissues was determined by real-time PCR. The obtained results showed that salsolinol infused at the higher dose significantly (p < 0.001) increased plasma prolactin concentration in lactating ewes, when compared with the concentration noted before the infusion and with that in lactating controls. In lactating ewes, the relative levels of prolactin mRNA expression in the AP and MBH were up to twofold and fivefold higher respectively than in non-lactating ewes (p < 0.05). In our experimental design, salsolinol did not significantly affect the ongoing process of prolactin gene expression in these tissues. We conclude that in ewes, salsolinol may be involved, at least, in the process of stimulation of prolactin release during lactation and that hypothalamic prolactin plays an important role in the central mechanisms of adaptation to lactation.