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.

Effect of Neurosteroids on Basal and Stress-Induced Oxytocin Secretion in Luteal-Phase and Pregnant Sheep.

Oxytocin (OT) is a neuropeptide synthesized in the hypothalamic nuclei that modulates both behavioral and reproductive functions, associated with the increased neurosteroid synthesis in the brain. Therefore, the present study tested the hypothesis that manipulation of central neurosteroid levels could affect oxytocin synthesis and release in non-pregnant and pregnant sheep under both basal and stressful conditions. In Experiment 1, luteal-phase sheep were subjected to a series of intracerebroventricular (icv.) infusions of allopregnanolone (AL, 4 × 15 µg/60 µL/30 min) for 3 days. In Experiment 2, pregnant animals (4th month) received a series of infusions of the neurosteroid synthesis blocker, finasteride (4 × 25 µg/60 µL/30 min), conducted for 3 days. In non-pregnant sheep AL alone was shown to differentially modulate OT synthesis in basal conditions, and strongly inhibit OT response to stress (p < 0.001). In contrast, in pregnant animals, basal and stress-induced OT secretion was significantly (p < 0.001) increased during finasteride infusion compared to controls. In conclusion, we showed that neurosteroids were involved in the control of OT secretion in sheep, particularly under stress and pregnancy conditions and are part of an adaptive mechanism which is responsible for protecting and maintaining pregnancy in harmful situations.

Involvement of neurosteroids in the control of prolactin secretion in sheep under basal, stressful and pregnancy conditions.

Prolactin (PRL) secretion by the anterior pituitary (AP) is responsive to changes in physiological conditions and many external factors that also affect brain neurosteroid levels. This study tested the hypothesis that neurosteroids can affect PRL secretion in sheep under basal, stressful and advanced pregnancy conditions. In Experiment 1, luteal-phase sheep were subjected to a three-day series of intracerebroventricular (icv.) control (n = 12) or allopregnanolone (AL, 4 × 15 µg/60 µL/30 min at 30-min intervals, n = 12) infusions. Acute stressful stimuli, isolation and partial movement restriction were applied on the third day of infusion to half of the animals in each group. In Experiment 2, pregnant sheep were subjected to a three-day series of icv. control (n = 6) or finasteride (4 × 25 µg/60 µL/30 min at 30-min intervals, n = 6) infusions during the 16th week of pregnancy. As a result, the relative abundance of PRL transcript increased in the AP of luteal-phase sheep treated with stress, AL and AL in combination with stress (P < 0.05 - P < 0.01) compared to controls. The level of PRL mRNA in stressed-AL-treated sheep was higher (P < 0.01) than in sheep only subjected to stress. The PRL protein content in the AP decreased in stressed-only sheep compared to controls (P < 0.05) and increased in stressed-AL-treated sheep compared to controls and other groups (P < 0.05 - P < 0.01). Plasma PRL concentration increased (P < 0.05 - P < 0.01) in stressed-only sheep compared to controls; AL infusion counteracted the stress-induced increase in PRL levels (P < 0.05 - P < 0.01) and had no effect in non-stressed animals. Inhibition of neurosteroid synthesis in the brain of pregnant sheep by finasteride caused transient increases (P < 0.05 - P < 0.001) in plasma PRL concentration compared to controls. In conclusion, the presented results indicate a bimodal effect of AL on PRL secretion in sheep: first at the molecular level - stimulation of PRL mRNA expression; second - inhibition of hormone release from pituitary lactotrophs. Both AL activities may involve various mechanisms regulating PRL secretion. In general, cerebral neurosteroids can affect the supply of pituitary PRL in the body under certain conditions, such as stress and pregnancy.

Central Stimulatory Effect of Kynurenic Acid on BDNF-TrkB Signaling and BER Enzymatic Activity in the Hippocampal CA1 Field in Sheep.

Deficiency of neurotrophic factors and oxidative DNA damage are common causes of many neurodegenerative diseases. Recently, the importance of kynurenic acid (KYNA), an active metabolite of tryptophan, has increased as a neuroprotective molecule in the brain. Therefore, the present study tested the hypothesis that centrally acting KYNA would positively affect: (1) brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling and (2) selected base excision repair (BER) pathway enzymes activities in the hippocampal CA1 field in sheep. Both lower (20 µg in total) and higher (100 µg in total) doses of KYNA infused into the third brain ventricle differentially increased the abundance of BDNF and TrkB mRNA in the CA1 field; additionally, the higher dose increased BDNF tissue concentration. The lower dose of KYNA increased mRNA expression for 8-oxoguanine glycosylase (OGG1), N-methylpurine DNA glycosylase (MPG), and thymine DNA glycosylase and stimulated the repair of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxy-cytosine as determined by the excision efficiency of lesioned nucleobases. The higher dose increased the abundance of OGG1 and MPG transcripts, however, its stimulatory effect on repair activity was less pronounced in all cases compared to the lower dose. The increased level of AP-endonuclease mRNA expression was dose-dependent. In conclusion, the potential neurotrophic and neuroprotective effects of KYNA in brain cells may involve stimulation of the BDNF-TrkB and BER pathways.

Segmentation of Preretinal Space in Optical Coherence Tomography Images Using Deep Neural Networks.

This paper proposes an efficient segmentation of the preretinal area between the inner limiting membrane (ILM) and posterior cortical vitreous (PCV) of the human eye in an image obtained with the use of optical coherence tomography (OCT). The research was carried out using a database of three-dimensional OCT imaging scans obtained with the Optovue RTVue XR Avanti device. Various types of neural networks (UNet, Attention UNet, ReLayNet, LFUNet) were tested for semantic segmentation, their effectiveness was assessed using the Dice coefficient and compared to the graph theory techniques. Improvement in segmentation efficiency was achieved through the use of relative distance maps. We also show that selecting a larger kernel size for convolutional layers can improve segmentation quality depending on the neural network model. In the case of PVC, we obtain the effectiveness reaching up to 96.35%. The proposed solution can be widely used to diagnose vitreomacular traction changes, which is not yet available in scientific or commercial OCT imaging solutions.

Involvement of neurosteroids in the control of hypothalamic-pituitary-adrenal axis activity in pregnant sheep under basal and stressful conditions.

Neurosteroids are synthesized locally in the brain, where they can modify neuronal functionality depending on the physiological state. A high correlation was demonstrated between the increasing activity of the hypothalamic-pituitary-adrenal (HPA) axis and allopregnanolone (AL) concentration in the cerebrospinal fluid in sheep during pregnancy. Therefore, the present study tested the hypothesis that blocking neurosteroid synthesis in the brain of a pregnant sheep would affect HPA axis activity under both basal and stressful conditions. Two groups of sheep in the fourth month of gestation (n = 7 each) were subjected to the following treatments: 1) intracerebroventricular (icv) infusion of vehicle for three days (C) and then icv infusion of finasteride (a total of 100 µg/240 µL/day) for three days (F), one week apart, and 2) icv infusion of vehicle for three days and application of stressful stimuli (isolation and partial movement restriction) on the third day (S), and subsequently icv infusion of finasteride for three days and application of stressful stimuli on the third day (SF), one week apart. On the third days of the experiment, a 4-h push-pull perfusion of the infundibular nucleus/median eminence and blood sampling were performed. Mean perfusate corticotropin-releasing hormone (CRH), plasma adrenocorticotropin (ACTH) and cortisol concentrations were significantly higher in sheep treated with finasteride, stress and finasteride in combination with stress compared to controls. The highest hormone concentrations in Groups F, S and SF, were recorded during the first 60 min; however, significant increases in CRH and ACTH levels were observed in Group SF towards the end of the experiment. It can be concluded that neurosteroids may be an essential component of the mechanism controlling HPA axis activity in pregnant sheep, not only under stress-free conditions, but more importantly, also by inhibiting the neuroendocrine response to stressors.

The Effect of Allopregnanolone on Enzymatic Activity of the DNA Base Excision Repair Pathway in the Sheep Hippocampus and Amygdala under Natural and Stressful Conditions.

The neurosteroid allopregnanolone (AL) has many beneficial functions in the brain. This study tested the hypothesis that AL administered for three days into the third brain ventricle would affect the enzymatic activity of the DNA base excision repair (BER) pathway in the hippocampal CA1 and CA3 fields and the central amygdala in luteal-phase sheep under both natural and stressful conditions. Acute stressful stimuli, including isolation and partial movement restriction, were used on the last day of infusion. The results showed that stressful stimuli increased N-methylpurine DNA glycosylase (MPG), thymine DNA glycosylase (TDG), 8-oxoguanine glycosylase (OGG1), and AP-endonuclease 1 (APE1) mRNA expression, as well as repair activities for 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC), and 8-oxoguanine (8-oxoG) compared to controls. The stimulated events were lower in stressed and AL-treated sheep compared to sheep that were only stressed (except MPG mRNA expression in the CA1 and amygdala, as well as TDG mRNA expression in the CA1). AL alone reduced mRNA expression of all DNA repair enzymes (except TDG in the amygdala) relative to controls and other groups. DNA repair activities varied depending on the tissue-AL alone stimulated the excision of εA in the amygdala, εC in the CA3 and amygdala, and 8-oxoG in all tissues studied compared to controls. However, the excision efficiency of lesioned bases in the AL group was lower than in the stressed and stressed and AL-treated groups, with the exception of εA in the amygdala. In conclusion, the presented modulating effect of AL on the synthesis of BER pathway enzymes and their repair capacity, both under natural and stressful conditions, indicates another functional role of this neurosteroid in brain structures.

Effects of allopregnanolone on central reproductive functions in sheep under natural and stressful conditions.

Reproductive functions may be affected by internal and external factors that are integrated in the central nervous system (CNS). Stressful stimuli induce the neuroendocrine response of the hypothalamic-pituitary-adrenal axis, as well as the synthesis of the neurosteroid allopregnanolone (AL) in the brain. This study tested the hypothesis that centrally administered AL could affect the expression of certain genes involved in reproductive functions at the hypothalamus and pituitary levels, as well as pulsatile gonadotropin secretion in sheep under both natural and stressful conditions. Luteal-phase sheep (n = 24) were subjected to a three-day (day 12-14 of the estrous cycle) series of control or AL (4 × 15 µg/60 µL/30 min, at 30 min intervals) infusions into the third ventricle. Acute stressful stimuli (isolation from other sheep and partial movement restriction) were used in the third day of infusion. Stressful stimuli reduced kisspeptin-1 mRNA levels in both the mediobasal hypothalamus (MBH) and the preoptic area (POA), while pro-dynorphin (PDYN) mRNA level only in the MBH. AL alone decreased the abundances of these transcripts in both structures. Stress increased the expression of gonadotropin-releasing hormone (GnRH) mRNA in the MBH and POA, luteinizing hormone (LH) ß subunit (LHß) mRNA in the anterior pituitary (AP) and pulsatile LH secretion. In contrast, mRNA level of follicle stimulating hormone (FSH) ß subunit (FSHß) was decreased in the AP, with no effect of stress on pulsatile FSH secretion. In stressed sheep, AL counteracted the increase in GnRH mRNA expression only in the POA, but it decreased the level of this transcript in both hypothalamic tissues when infused alone. AL prevented the stress-induced increase in LHß mRNA expression in the AP and pulsatile LH secretion, as well as inhibited almost all aspects of FSH secretion when administered alone. The suppressive effect of AL on GnRH receptor mRNA expression was also observed in both MBH and AP. We concluded that acute stress and AL exerted multidirectional effects on hypothalamic centers that regulate reproductive functions and secretory activity of AP gonadotrophs in sheep. However, we indicated the dominant inhibitory effect of AL under natural and stressful conditions.

Allopregnanolone reduces neuroendocrine response to acute stressful stimuli in sheep.

The verified hypothesis assumed that centrally administered neurosteroid, allopregnanolone (AL), could affect basal and/or stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis in sheep. Four groups (n = 6 each) of luteal-phase sheep were intracerebroventricularly infused for 3 days with a vehicle without stress (control); a vehicle treated with stressful stimuli (isolation and partial movement restriction) on the third day; AL (4 × 15 µg/60 µL/30 min, at 30-min intervals) treated with stressful stimuli, and AL alone. Simultaneously, the push-pull perfusion of the infundibular nucleus/median eminence and plasma sample collection were performed. After the experiment, the sheep were killed to collect the hypothalamic and anterior pituitary (AP) tissues. Stressful stimuli evoked an increase in the expression of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the hypothalamic paraventricular nucleus (PVN), and AVP receptor (V1b) and proopiomelanocortin (POMC) mRNA in the AP; the concentrations of perfusate CRH, and plasma adrenocorticotropic hormone (ACTH) and cortisol compared to controls. Conversely, the expression of the CRH receptor (CRHR1) mRNA in the AP was downregulated. AL decreased the expression of CRH and AVP mRNA in the PVN, and AVPRV1b and POMC mRNA in the AP in stressed sheep, compared to only stressed ones. There was also a reduction in perfusate CRH, and plasma ACTH and cortisol concentrations. AL alone decreased the expression of CRHR1 mRNA in the AP, and plasma cortisol concentration at the beginning of the collection period compared to controls. In conclusion, AL may function centrally as a suppressor of HPA axis activity in stressed sheep.

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.

IMAGING AND MEASUREMENT OF THE PRERETINAL SPACE IN VITREOMACULAR ADHESION AND VITREOMACULAR TRACTION BY A NEW SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY ANALYSIS.

PURPOSE: To evaluate a new method for volumetric imaging of the preretinal space (also known as the subhyaloid, subcortical, or retrocortical space) and investigate differences in preretinal space volume in vitreomacular adhesion (VMA) and vitreomacular traction (VMT). METHODS: Nine patients with VMA and 13 with VMT were prospectively evaluated. Automatic inner limiting membrane line segmentation, which exploits graph search theory implementation, and posterior cortical vitreous line segmentation were performed on 141 horizontal spectral domain optical coherence tomography B-scans per patient. Vertical distances (depths) between the posterior cortical vitreous and inner limiting membrane lines were calculated for each optical coherence tomography B-scan acquired. The derived distances were merged and visualized as a color depth map that represented the preretinal space between the posterior surface of the hyaloid and the anterior surface of the retina. The early treatment d retinopathy study macular map was overlaid onto final virtual maps, and preretinal space volumes were calculated for each early treatment diabetic retinopathy study map sector. RESULTS: Volumetric maps representing preretinal space volumes were created for each patient in the VMA and VMT groups. Preretinal space volumes were larger in all early treatment diabetic retinopathy study map macular regions in the VMT group compared with those in the VMA group. The differences reached statistical significance in all early treatment diabetic retinopathy study sectors, except for the superior outer macula and temporal outer macula where significance values were P = 0.05 and P = 0.08, respectively. Overall, the relative differences in preretinal space volumes between the VMT and VMA groups varied from 2.7 to 4.3 in inner regions and 1.8 to 2.9 in outer regions. CONCLUSION: Our study provides evidence of significant differences in preretinal space volume between eyes with VMA and those with VMT. This may be useful not only in the investigation of preretinal space properties in VMA and VMT, but also in other conditions, such as age-related macular degeneration, diabetic retinopathy, and central retinal vein occlusion.

Hypothalamic-pituitary GnRH/LH axis activity is affected by salsolinol in sheep during lactation: Effects of intracerebroventricular infusions of salsolinol and its antagonizing analogue.

The aim of the study was to test the hypothesis that salsolinol, a derivative of dopamine, is involved in the regulation of hypothalamic-pituitary gonadotropic (GnRH/LH) axis activity in lactating sheep. In the first experiment performed on sheep during the fifth week of lactation, a structural analogue of salsolinol (1-MeDIQ) was infused into the third brain ventricle (IIIv) to antagonize its action within the central nervous system (CNS). A push-pull perfusion of the infundibular nucleus/median eminence was performed simultaneously, and blood samples were collected from the jugular vein. In the second experiment, sheep received infusions of salsolinol into the IIIv, 48 hours after the weaning of their 8-week-old lambs. Blood samples were collected during the experimental periods, and the anterior pituitary (AP) tissue was dissected immediately after the end of the experiment. Perfusate GnRH concentration (experiment 1), plasma LH concentration (experiments 1 and 2), and relative LHß mRNA levels in the AP tissue (experiment 2) were assayed. Blocking of salsolinol action in the CNS of lactating sheep caused a significant (P < 0.001) decrease in the perfusate GnRH concentrations in comparison with controls. Treatment with 1-MEDIQ also significantly decreased (P < 0.001) the LH concentration in the blood plasma. In turn, salsolinol infused 48 hours after lamb weaning significantly (P < 0.001) increased plasma LH concentration, reflected in the significant (P < 0.05) increase in the amplitude of LH pulses in the treated sheep as compared to the control animals. There was no significant difference in the relative levels of LHß-subunit mRNA in the AP between control and salsolinol-infused sheep. The results lead to a conclusion that salsolinol affects the secretory activity of the GnRH/LH axis in sheep during lactation. Whether salsolinol infused into the IIIv evokes this stimulatory effect by itself or by modulation of other regulatory systems needs to be clarified.