Long-term OVX Rats Research Articles

Different Effects of Fluoxetine and Paroxetine Combined with Vitamin D3 in Ovariectomized Rats Exposed to Unpredictable Stress

Background: Vitamin D3 (VD3) is involved in the pathophysiological mechanisms of affective-related disorders and controls the functional activity of various hormonal systems. The complex interaction between estrogen and VD3 creates a neurobiological basis for their participation in similar behavioral disorders. Objectives: This study aimed to evaluate whether VD3 (5.0 mg/kg, s.c.) facilitates the antidepressant-like action of fluoxetine (10.0 mg/kg, i.p.) or paroxetine (10.0 mg/kg, i.p.) by enhancing the antidepressant-like activity of these drugs in adult long-term Ovariectomized (OVX) rats subjected to Chronic Unpredictable Mild Stress (CUMS) protocol for 6 weeks. Methods: Sucrose Preference (SPT) and Forced Swim (FST) tests were performed to evaluate the anhedonia state and depressive symptoms, respectively. The Open-Field Test (OFT) was carried out to measure locomotor activity as well as grooming behavior produced by CUMS in long-term OVX rats. Corticosterone (CS)/estradiol (E2) in the serum was tested by rat ELISA kits. NF-kB, 5-HT/5-HIIA, and pro-inflammatory cytokine levels in the hippocampus were also examined by rat ELISA kits. Results: The results of this study suggest that combined treatment with fluoxetine (10.0 mg/kg, i.p.) or paroxetine (10.0 mg/kg, i.p.) along with VD3 (5.0 mg/kg, s.c.) produces distinct effects on the depression-like behavior in long-term OVX/CUMS rats. Co-administration of fluoxetine (10.0 mg/kg, i.p.) with VD3 did not facilitate the antidepressant-like effects of fluoxetine in the long-term OVX rats with CUMS. On the other hand, co-treatment with paroxetine with VD3 resulted in faster and marked antianhedonic- and antidepressant-like effects in long-term OVX rats with CUMS as compared to treatment with paroxetine alone. The co-administration of paroxetine and VD3 attenuates stress-induced modifications of CS/E2 levels in the serum, as well as- proinflammatory cytokine/NF-kB/5-HT levels in the hippocampus of long-term OVX rats exposed to CUMS. Conclusion: Supplementation of VD3 (5.0 mg/kg, s.c.)to paroxetine (10.0 mg/kg, i.p.) facilitates antianhedonic- and antidepressant-like effects of paroxetine in adult long-term OVX rats exposed to CUMS.

Antidepressant activity of \u03c9-3 polyunsaturated fatty acids in ovariectomized rats: role of neuroinflammation and microglial polarization

BackgroundMenopause predisposes individuals to affective disorders, such as depression, which is tightly related to neuroinflammation. While the neuroinflammatory condition has been demonstrated in ovariectomized (OVX) rodents, there is limited evidence concerning microglial polarization, a key process in brain immune activation, in menopause-related brain.MethodsTherefore, the present study aims to evaluate the polarized microglia in long-term OVX rats and we further explored whether supplementation of ω-3 polyunsaturated fatty acids (PUFA), the pleiotropic bioactive nutrient, is effective in the neurobehavioral changes caused by OVX.ResultsOur data showed that OVX-induced anxiety and depression-like behaviors in rats, accompanied with increased neural apoptosis and microglial activation in the hippocampus. Additionally, OVX enhanced proinflammatory cytokines expression and suppressed the expression of anti-inflammatory cytokine, IL-10. Correspondingly, OVX reinforced NFκB signaling and shifted the microglia from immunoregulatory M2 phenotype to proinflammatory M1 phenotype. Meanwhile, daily supplementation with PUFA suppressed microglial M1 polarization and potentiated M2 polarization in OVX rats. In parallel, PUFA also exerted antidepressant and neuroprotective activities, accompanied with neuroimmune-modulating actions.ConclusionCollectively, the present study firstly demonstrated the disturbed microglial polarization in the OVX brain and provide novel evidence showing the association between the antidepressant actions of PUFA and the restraint neuroinflammatory progression.

Effects of Vitamin D3 in Long-Term Ovariectomized Rats Subjected to Chronic Unpredictable Mild Stress: BDNF, NT-3, and NT-4 Implications.

The purpose of this study was to explore the antidepressant-like effects of vitamin D3 at different doses (1.0, 2.5, and 5.0 mg/kg sc) on a model of depression produced by chronic unpredictable mild stress (CUMS) for 28 days in long-term (3 months) ovariectomized (OVX) adult rats. Sucrose preference (SPT), forced swimming (FST) and open-field (OFT) tests were conducted to examine the depression-like state. Serum corticosterone/adrenocorticotrophic hormone (ACTH) levels and hippocampal brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3/NT-4 expressions by ELISA kits and/or western blotting were determined to assess the possible mechanisms of the vitamin D3 effects on the depression-like profile in long-term OVX rats subjected to CUMS. The results showed that vitamin D3 (5.0 mg/kg), as well as fluoxetine treatment, considerably reversed the depression-like state in the SPT and FST, decreased serum corticosterone/ACTH levels, and increased BDNF and NT-3/NT-4 levels in the hippocampus of long-term OVX rats compared to OVX rats with CUMS (p < 0.05). Thus, a high dose of vitamin D3 (5.0 mg/kg sc) could improve the depression-like profile in long-term OVX adult female rats subjected to the CUMS procedure, which might be mediated by the regulation of BDNF and the NT-3/NT-4 signaling pathways in the hippocampus, as well as the corticosterone/ACTH levels of the blood serum.

Swimming improves cognitive reserve in ovariectomized rats and enhances neuroprotection after global cerebral ischemia

Cognitive reserve has been proposed to account for different responses to brain damage or pathology. Factors implicated to influence cognitive reserve include cognitive engagement, physical activity, leisure activities, stress levels, and diet. Furthermore, long-term ovariectomy (OVX), such as occurs in women that have underwent surgical menopause, has been reported to increase the risk of cognitive impairment. In the current study, we examined whether swimming improves cognitive function in long-term OVX-rats. We also examined the neuroprotective effect of swimming after global cerebral ischemia (GCI) and explored the effect of swimming preconditioning on activation of the MAPK cascade signaling pathway, synaptic proteins and brain-derived growth factor (BDNF) – all factors implicated in regulating synaptic plasticity and neuroprotection in the brain. Adult Sprague–Dawley OVX-rats were randomly assigned into four groups: Sham (Sh), Sham + Swimming (Sh + Sw), Ischemia/Reperfusion (IR) and IR + Sw. Our results revealed that (1) Morris water maze and shuttle box test analysis revealed that swimming improved cognitive function in OVX-rats, (2) The levels of PSD95 and synaptophysin, as well as the protein expression of p-ERK, p-CREB and BDNF were all increased in the hippocampus after swimming with or without GCI, and (3) Swimming also increased the number of surviving neurons and IL4 protein expression, while decreasing the Iba1 (a microglia marker) level in the hippocampus. In conclusion, our study demonstrates that swimming improves memory in OVX-rats, and that swimming preconditioning enhances the neuroprotective ERK1/2/CREB/BDNF pathway signaling and ameliorates brain damage after GCI in OVX-rats, which may be closely related to induction of an IL4-mediated anti-inflammatory mechanism.

Red clover Trifolium pratense (Linn.) isoflavones extract on the pain threshold of normal and ovariectomized rats – a long‐term study

Depletion of estrogens occurs in women during menopause, while in experimental animals, oophorectomy is a common method to deplete the animals of their gonadal hormones. Recently, phytoestrogens derived from plants have been tried as estrogen substitutes during menopause. In the present study an isoflavones methanol extract from red clover Trifolium pratense (Linn.) was administered orally (500 mg/kg of body weight) to ovariectomized (OVX) and normal (controls) rats for 90 and 180 days. Their pain threshold was monitored using tail flicking and formalin test methods. Observations showed that the OVX rat pain threshold was reduced due to estrogen deprivation, whereas the pain threshold levels in OVX rats treated with isoflavones extract was similar to the control animals. The present study demonstrated the influence of phytoestrogen on long-term OVX rats in pain perception in the absence of ovarian estrogen and without toxic side effects. However, the actions of gonadal hormones on nociceptive axis are myriad and complex, so further studies on the exact physiological mechanism of the phytoestrogen action on nociceptive axis is warranted.

Non-steroidal factors in bovine follicular fluid inhibit or facilitate the action of pulsatile administration of GnRH on LH release in the female rat.

Using steroid-free bovine follicular fluid (bFF), we studied the action of gonadotrophin surge-inhibiting factor/attenuating factor (GnSIF/AF) on GnRH-induced self-priming in phenobarbital-blocked female rats. For the experiments we used intact rats, short-term (4 h) ovariectomized (OVX) rats and long-term (14 days) OVX rats. In the latter case the rats were injected with 17beta-oestradiol benzoate (OB, 40 micrograms) or vehicle only, 2 or 48 h before the experiment. GnRH (10-50 pmol/kg body weight) was injected intra-arterially in 5 or 15 pulses, respectively 60 or 20 min apart, starting 1 or 4 h after injection of bFF (0.5 or 1.0 ml). In response to 25 pmol/kg GnRH pulses (1/h), we observed no effect in the long-term OVX rats, a minor effect in the intact rats and an enhanced self-priming effect in the short-term OVX rats. Administration of bFF attenuated or even completely inhibited the self-priming process. However, in the case of long-term OVX rats LH release was inhibited only after long-term OB priming. Furthermore, 4 h after administration of bFF, LH release in response to 25 pmol/kg GnRH pulses (3/h) was inhibited transiently in intact rats and long-term OVX rats. The results support the hypothesis of a functional antagonistic action between GnRH and GnSIF/AF. However, when injected 1 h before, bFF facilitated the initial release of the surge-like LH pattern in intact rats in response to 3 pulses/h of GnRH. These results are consistent with an important role of GnSIF/AF and other non-steroidal ovarian factors in the control of both low LH concentrations and the generation of the LH surge. Some genomic action of oestradiol might be a prerequisite for the inhibitory effect of GnSIF/AF on GnRH-induced LH release.

Circadian changes of serum prolactin levels and tuberoinfundibular dopaminergic neuron activities in ovariectomized rats treated with or without estrogen: the role of the suprachiasmatic nuclei.

Variations of serum prolactin (PRL) levels and activities of tuberoinfundibular dopaminergic (TIDA) neurons during the afternoon of ovariectomized (OVX) rats treated with or without estrogen were determined in this study. Long-term OVX rats treated with or without polyestradiol phosphate (0.1 mg/rat, s.c.) were decapitated every hour from 10.00 to 19.00 h (except 11.00 and 13.00 h). Serum PRL and median eminence (ME) dihydroxyphenylacetic acid (DOPAC) or dihydroxyphenylalanine (DOPA) levels were determined by radioimmunoassay and high performance liquid chromatography plus electrochemical detection, respectively. A prominent PRL surge started and peaked around 14.00-15.00 h, and remained significantly higher than levels of 10.00 and 12.00 h throughout the afternoon. Significant decreases of ME DOPAC and DOPA concentrations were also observed between 14.00 and 19.00 h. In OVX rats with no estrogen replacement, no PRL surge was observed and the changes of ME DOPAC concentrations during the afternoon were not significant except for that at 17.00 h. The ME DOPA accumulation, however, exhibited significantly lower levels from 14.00 to 19.00 h than that at 12.00 h, indicating that an endogenous rhythm for DA synthesis existed in OVX rats. In estrogen-treated OVX rats bearing bilateral lesions of the suprachiasmatic nuclei, both changes in serum PRL level and TIDA neuron activity were abolished. We conclude that an endogenous rhythm of the activities of TIDA neurons may exist in both OVX and OVX plus estrogen-treated rats. The rhythm is regulated by the suprachiasmatic nuclei and may be amplified by estrogen for the induction of PRL surge.

Evidence that noradrenergic activity does not mediate the inhibition of luteinizing hormone secretion caused by vasoactive intestinal peptide

We have recently shown that intracerebroventricular (i.c.v.) infusion of vasoactive intestinal peptide (VIP) in ovariectomized (OVX) rats causes a pronounced inhibition of pulsatile luteinizing (LH) secretion. Since there is increasing evidence for an interaction between VIP- and norepinephrine-secreting systems in the brain, we tested the hypothesis that disruption of central noradrenergic activity would attenuate VIP-induced suppression of pulsatile LH release. Seven days prior to blood sampling, we administered either 6-hydroxydopamine (30 μg) or vehicle into the third cerebral ventricle of long-term OVX rats. This treatment reduced hypothalamic norepinephrine concentrations to 36% of control values. On the day of blood sampling, serial 300-μl blood samples were taken from conscious, unrestrained animals at 5-min intercals for a tota; of 4 h. Each sample was replaced with an equal volume of a blood replacement mixture. Immediately following a control bleeding period of 2 h, VIP was administered i.c.v. to each animal for 2 h (at a dose of 1.8 nmol/h). The suppression of pulsatile LH secretion caused by VIP infusion was virtually identical in the 6-hydroxydopamine- and vehicle-pretreated groups. These results indicate that a substantial depletion of hypothalamic norepinephrine does not attenuate VIP-induced suppression of pulsatile LH secretion. This provides evidence that centrally administered VIP inhibits pulsatile LH secretion by a mechanism independent of noradrenergic transmission.

Norepinephrine inhibition of pulsatile LH release: receptor specificity.

Infusion of norepinephrine (NE) into the third ventricle of ovariectomized (OVX) adult rats inhibits pulsatile luteinizing hormone (LH) secretion. This study examines the effects of pretreatment with specific alpha- and beta-adrenergic blockers on the subsequent NE-induced suppression of LH release. Unanesthetized long-term OVX rats were injected ip or iv with phenoxybenzamine (alpha-adrenergic receptor blocker), propranolol (beta-adrenergic blocker), or an equal volume of saline (as controls). Approximately 1-1.5 h later, the animals were given an intraventricular (ivt) administration of NE. Infusion of acidified saline into the third ventricle of OVX rats failed to affect the pulsatile pattern of LH release characteristic of these animals. After ivt of NE, the control rats showed a significant decrease in mean blood LH level of ca. 30%. Pretreatment of OVX rat with propranolol either ivt or ip failed to affect pulsatile LH release. Subsequent ivt infusion of NE either at 1 h or 15 min after propranolol treatment also caused a 28-30% decrease in the mean LH levels and suppressed pulsatile discharge of LH, an effect that lasted for approximately 45-50 min. In sharp contrast to the saline and propranolol-treated groups, rats pretreated with phenoxybenzamine had sporadic or no LH pulses during the 70-min postphenoxybenzamine period and had significantly lower mean LH levels when compared with control animals (P less than 0.05). Moreover, a drop in mean blood LH levels after NE infusion was not observed in the rats pretreated with phenoxybenzamine.(ABSTRACT TRUNCATED AT 250 WORDS)

A POSSIBLE ROLE OF CORPORA LUTEA WITH REGARD TO OESTROGEN-INDUCED CHANGES IN PITUITARY RESPONSIVENESS TO LH-RH

The effect of oestradiol benzoate (OeB; 3 micrograms sc) on LH-RH-induced LH-secretory responses was investigated in: (1) long-term ovariectomized (OVX) rats; (2) intact (i.e. sham-OVX) and 24 h-OVX persistent oestrous (PO) rats (intact PO rats do not possess corpora lutea); (3) intact and 24 h-OVX cyclic (dioestrous) rats (intact cyclic rats possess "not-activated" corpora lutea), and (4) intact and 24 h-OVX phase-2-pseudopregnant (phase-2-PSP) rats (phase 2 of PSP lasts from day 2 to day 10. Experiments were performed on days 7--8. Intact phase-2-PSP rats possess "activated" corpora lutea). A "positive" effect of OeB was observed in: (1) long-term OVX rats; (2) intact and 24 h-OVX PO rats; (3) 24 h-OVX cyclic rats and (4) 24 h-OVX phase-2-PSP rats. In intact cyclic and phase-2-PSP rats OeB was completely ineffective. From these observations it is concluded that only in the absence of corpora lutea OeB can exert a "positive" effect on LH-RH-induced LH-secretory responses. Ovariectomy alone, however, may have a negative impact on LH-RH-induced LH-secretory responses. This holds for PO- and phase-3-PSP rats (phase 3 of PSP begins once the corpora lutea come to lysis, i.e. on or shortly before day 10, and lasts until the end of PSP), but not for phase-2-PSP rats. In cyclic rats, on the other hand, ovariectomy performed on dioestrus-1 "only" interferes with the normal development of pituitary responsiveness to LH-RH and this can be nullified by OeB. Our observations suggest that (1) relatively long-acting products of corpora lutea "stabilize" (phase-2-PSP rats), c.q. limit the (almost certainly oestrogen-dependent) development of pituitary responsiveness to LH-RH (cyclic and phase-3-PSP rats); (2) in ovariectomized rats the influence of these products vanishes and that (3) this results in a situation in which OeB can become effective. It is also suggested that in the course of the ovulatory cycle and phase 3 of PSP a similar disappearance of corpora luteal products occurs "spontaneously" as a result of the process of luteolysis with as a consequence that the pituitary responsiveness to LH-RH, under the influence of endogenous oestrogens, develops continuously until, on pro-oestrus, an LH-surge occurs. Evidence is presented that the corpora luteal products, postulated in this paper, are not identical to progesterone.

A Medial Basal Hypothalamic Site of Synergistic Action of Estrogen and Progesterone on the Inhibition of Pituitary Luteinizing Hormone Release1

Plasma LH concentrations were measured by radioimmunoassay in blood withdrawn from indwelling atrial cannulas in long-term ovariectomized (OVX) rats. Subcutaneous injection of 50 μg of estradiol benzoate (EB) in oil depressed the plasma LH concentration to about one-third within 2 h. In other rats, sc injection of 25 mg of progesterone (P) in oil at the time of EB injection did not alter this response. Plasma LH remained suppressed at that level until at least 8 h after injection in both groups, but fell an additional 50% within 24 h after injection only in the rats treated with both EB and P. In additional rats, P, but not the oil vehicle alone, when administered 18 h after the injection of EB, caused plasma LH to decline by about 50% within 60 min. Under these latter conditions, 5, 20 or 100 ng of LHRH was injected through the cannula at 1 h after the injection of P or oil. Progesterone did not inhibit pituitary LH release in response to LHRH as determined by comparison of plasma LH concentrations and increments at 3, 10 and 30 min after LHRH in the two groups. In OVX rats which still exhibited pulsatile LH release after surgical interruption of all of the neural connections to their medial basal hypothalami, injection of EB followed 18 h later by injection of P produced results similar to those seen in control OVX rats. The results suggest that P acts synergistically with estrogen at the level of the medial basal hypothalamus in long-term OVX rats to lower plasma LH concentrations to levels which approach but do not reach basal plasma LH levels in cyclic rats and that, under the conditions of this study, P does not lower plasma LH concentration in EB-treated OVX rats until estrogen has exerted its effect for >8 h but ≤18 h.

Pulsatile discharges of luteinizing hormone in the ovariectomized rat during the 24-hour day.

Blood samples were taken every 5 min for a 2-h period commencing at 09.00 h or epsilon 13.00 h from long-term ovariectomized (OVX) rats under regular lighting conditions (lights on 05.00-19.00 h), and commencing at 09.30 h or 14.00 h from long-term OVX rats under reversed lighting conditions (lights on 18.00-08.00 h), the latter equivalent to starting sampling at 20.30 h or 01.00 h under regular lighting. Five rats were used in each group, and luteinizing hormone (LH) was measured in duplicate aliquots of each sample by radioimmunoassay. Pulsatile increases in plasma LH concentrations were observed in all 20 animals, and mean, peak, and nadir plasma LH concentrations for each group did not differ between sampling periods. Similarly, the time of day did not affect the frequency at which pulsatile discharges of LH occurred. These data suggest that pulsatile discharges of LH in long-term OVX rats occur throughout the 24-h day.