Middle-aged Female Rats Research Articles

Abstract WMP118: Neuroprotective Effects of IGF1 on Brain Microvascular Endothelial Cells Under Ischemic Conditions Can Be Replicated by Antagomirs to MicroRNA(Mir-92b) and Mir33a

Background: Insulin like growth factor (IGF1) treatment to middle aged female rats post stroke reduces infarct volume, and this is preceded by reduced permeability of the blood brain barrier (BBB) at 4h post stroke. Analysis of ischemic tissue at 4h showed that IGF1 treatment significantly reduced expression of 8 miRNA. To determine if these miRNA are mechanistically linked to barrier function, we tested the impact of these miRNA on barrier function using human brain microvascular endothelial cells (hBMECs) under ischemic conditions. Specifically, we mimicked the actions of IGF1 by using antagomirs to miR-29c, miR-33a and miR-92b. Methods: hBMECs were grown to confluence on collagen-coated dishes (5% CO2, 95% air) and exposed to acute oxygen-glucose deprivation (6h OGD, 1% O2 % and 5 % CO2) in the presence of IGF1 or vehicle. In addition, cultures were also treated with IGF1+JB-1 (IGFR antagonist), IGF1+ LY294002 (Akt inhibitor) and rapamycin (mTOR inhibitor), to establish IGF1 mediated pathways on barrier function. In parallel, cultures were exposed to antagomirs for mir29c, mir 33a and mir92b or scrambled oligo controls. Integrity of the cell monolayer was assessed by tomato-lectin staining and LDH assays for cell death. Lectin stained cell cultures (3 experimental replicates) were photographed, coded and analyzed for continuity of monolayer using a novel algorithm to determine intercellular ‘spaces’. Results: OGD caused significant retraction (11 fold increase, p<0.02) and discontinuous aggregates of cells, while IGF1 preserved the integrity of the endothelial monolayer and were no different from normoxic condition. JB-1 (13 fold increase,p<0.01) and Akt inhibitor (3.3 fold, p<0.02) abrogated the effects of IGF-1. Antagomirs to mir92b and mir33a also maintained cell integrity after OGD, (7 fold,p<0.03) while mir29c and scrambled oligos (10 fold damage, p<0.04) were ineffective. LDH assay was not significantly different in any of the treatment conditions, indicating that necrotic cell death was not altered. Conclusions: These findings provide direct evidence that IGF-1 actions at the endothelial barrier are replicated by specific miRNA and raise the possibility that IGF-1’s actions can be replicated by targeted suppression of miRNAs.

Sex- and age-associated differences in episodic-like memory and transcriptional regulation of hippocampal steroidogenic enzymes in rats.

The aim of this study was to evaluate the episodic-like memory (ELM) and the transcriptional regulation of the enzymes involved in hippocampal allopregnanolone synthesis in young adult and middle-aged male and female rats. Young adult males, but not middle-aged ones, showed a good performance in the ELM task. In contrast, neither young nor middle-aged females were able to discriminate the spatial order in which the objects were presented. In females, aging decreased the transcription of steroidogenic-related genes. In addition, the mRNA levels of 5α-reductase-1 were higher and the methylation of its promoter was lower in young adult females than in males, suggesting an epigenetic control. Further studies are needed to establish correlations between ELM and the transcriptional regulation of hippocampal steroidogenic enzymes. Our results contribute to the knowledge of sex differences in gene expression, methylation and memory during aging.

Intermittent fasting combined with supplementation with Ayurvedic herbs reduces anxiety in middle aged female rats by anti-inflammatory pathways.

Intermittent fasting-dietary restriction (IF-DR) is an increasingly popular intervention to promote healthy aging and delay age associated decline in brain functions. Also, the use of herbal interventions is gaining attention due to their non-pharmacological approach to treat several abnormalities and promote general health with least side effects. The present study was aimed to investigate the synergistic effects of IF-DR regimen with herbal supplementation on anxiety-like behavior and neuroinflammation in middle aged female rats. We used dried leaf powder of Withania somnifera and dried stem powder of Tinospora cordifolia for our study. The rats were divided into three groups: (1) Control group fed ad libitum (AL); (2) rats deprived of food for full day and fed ad libitum on every alternate day (IF-DR); and (3) IF-DR and herbal extract (DRH) group in which rats were fed ad libitum with herbal extract supplemented diet, every alternate day. Post regimen, the rats were tested for anxiety-like behavior and further used for study of key inflammatory molecules (NFκB, Iba1, TNFα, IL-1β, IL-6) and glial marker (GFAP) in hippocampus and piriform cortex regions of brain. The study was further extended to explore the effect of DRH regimen on stress response protein (HSP70) and calcium dependent regulators of synaptic plasticity (CaMKIIα, Calcineurin). Our data demonstrated that DRH regimen reduced anxiety-like behavior in middle age female rats and associated neuroinflammation by ameliorating key inflammatory cytokines and modulated stress response. The present data may provide scientific validation for anxiolytic and anti-inflammatory potential of herbal intervention combined with short term IF-DR regimen.

Soybean phytoestrogens reduce 27-hydroxycholesterol concentration in the liver of middle-aged female rats

Soybean phytoestrogens reduce 27-hydroxycholesterol concentration in the liver of middle-aged female rats

Sex Differences in Macrophage Functions in Middle-Aged Rats: Relevance of Estradiol Level and Macrophage Estrogen Receptor Expression.

The aim of this study was to examine the influence of sex on age-related changes in phenotype and functional capacity of rat macrophages. The potential role of estradiol as a contributing factor to a sex difference in macrophage function with age was also examined. Thioglycollate-elicited peritoneal macrophages derived from the young (2months old) and the naturally senescent intact middle-aged (16months old) male and female rats were tested for cytokine secretion and antimicrobial activity (NO and H2O2 production and myeloperoxidase activity). Serum concentration of estradiol and the expression of estrogen receptor (ER)α and ERβ on freshly isolated peritoneal macrophages were also examined. Decreased secretion of IL-1β and IL-6 by macrophages from middle-aged compared to the young females was accompanied with the lesser density of macrophage ERα expression and the lower systemic level of estradiol, whereas the opposite was true for middle-aged male rats. Macrophages in the middle-aged females, even with the diminished circulating estradiol levels, produce increased amount of IL-6, and comparable amounts of IL-1β, TNF-α, and NO to that measured in macrophages from the middle-aged males. Age-related changes in macrophage phenotype and the antimicrobial activity were independent of macrophage ERα/ERβ expression and estradiol level in both male and female rats. Although our study suggests that the sex difference in the level of circulating estradiol may to some extent contribute to sex difference in macrophage function of middle-aged rats, it also points to more complex hormonal regulation of peritoneal macrophage activity in females.

Effects of age and soybean isoflavones on hepatic cholesterol metabolism and thyroid hormone availability in acyclic female rats

Soy-food and its isoflavones, genistein (G) and daidzein (D), were reported to exert mild cholesterol-lowering effect, but the underlying mechanism is still unclear. In this research, first we studied age-related alterations in hepatic cholesterol metabolism of acyclic middle-aged (MA) female rats. Then we tested if purified isoflavones may prevent or reverse these changes, and whether putative changes in hepatic thyroid hormone availability may be associated with this effect. Serum and hepatic total cholesterol (TChol), bile acid and cholesterol precursors, as well as serum TSH and T4 concentrations, hepatic deiodinase (Dio) 1 enzyme activity and MCT8 protein expression were determined by comparing data obtained for MA with young adult (YA) intact (IC) females. Effects of subcutaneously administered G or D (35mg/kg) to MA rats were evaluated versus vehicle-treated MA females. MA IC females were characterized by: higher (p<0.05) serum TChol, lower (p<0.05) hepatic TChol and its biosynthetic precursors, lower (p<0.05) hepatic 7α-hydroxycholesterol but elevated (p<0.05) 27- and 24-hydroxycholesterol in comparison to YA IC. Both isoflavone treatments decreased (p<0.05) hepatic 27-hydroxycholesterol, G being more effective than D, without affecting any other parameter of Chol metabolism. Only G elevated hepatic Dio1 activity (p<0.05). In conclusion, age-related hypercholesteremia was associated with lower hepatic Chol synthesis and shift from main neutral (lower 7α-hydroxycholesterol) to alternative acidic pathway (higher 27-hydroxycholesterol) of Chol degradation to bile acid. Both isoflavones lowered hepatic 27-hydroxycholesterol, which may be considered beneficial. Only G treatment increased hepatic Dio1 activity, thus indicating local increase in thyroid hormones, obviously insufficient to induce prominent cholesterol-lowering effect.

Astrocyte-specific insulin-like growth factor-1 gene transfer in aging female rats improves stroke outcomes.

Middle aged female rats sustain larger stroke infarction and disability than younger female rats. This older group also shows age-related reduction of insulin like growth factor (IGF)-1 in serum and in astrocytes, a cell type necessary for poststroke recovery. To determine the impact of astrocytic IGF-1 for ischemic stroke, these studies tested the hypothesis that gene transfer of IGF-1 to astrocytes will improve stroke outcomes in middle aged female rats. Middle aged (10-12 month old), acyclic female rats were injected with recombinant adeno-associated virus serotype 5 (AAV5) packaged with the coding sequence of the human (h)IGF-1 gene downstream of an astrocyte-specific promoter glial fibrillary acidic protein (GFAP) (AAV5-GFP-hIGF-1) into the striatum and cortex. The AAV5-control consisted of an identical shuttle vector construct without the hIGF-1 gene (AAV5-GFAP-control). Six to eight weeks later, animals underwent transient (90 min) middle cerebral artery occlusion via intraluminal suture. While infarct volume was not altered, AAV5-GFAP-hIGF-1 treatment significantly improved blood pressure and neurological score in the early acute phase of stroke (2 days) and sensory-motor performance at both the early and late (5 days) acute phase of stroke. AAV5-GFAP-hIGF-1 treatment also reduced circulating serum levels of GFAP, a biomarker for blood brain barrier permeability. Flow cytometry analysis of immune cells in the brain at 24 hr poststroke showed that AAV5-GFAP-hIGF-1 altered the type of immune cells trafficked to the ischemic hemisphere, promoting an anti-inflammatory profile. Collectively, these studies show that targeted enhancement of IGF-1 in astrocytes of middle-aged females improves stroke-induced behavioral impairment and neuroinflammation.

Abstract WP107: Igf-1-mediated Neuroprotection for Ischemic Stroke is Preceded by Its Actions at the Endothelial Cell Barrier

Background: Our studies show that Insulin like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. We tested the hypothesis that IGF-1s neuroprotective effect is due to its action on the microvascular /endothelial barrier in the early acute phase of stroke. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal middle cerebral artery occlusion (MCAo) for 90 min followed by reperfusion and terminated 4h or 24h later. Animals received ICV infusion of artificial CSF, IGF-1 or IGF-1+JB-1. In separate experiments, we evaluated infarct volume, barrier assessment by uptake of Evan’s blue dye or histological evaluation of lectin stained microvessels. In parallel, cortical endothelial cells from middle-aged females were harvested and cultured ex vivo. Barrier properties were tested in cultures in transfer wells exposed to oxygen glucose deprivation (OGD) in the presence or absence of IGF-1 with FITC-labeled BSA in the media. Results: At 4h post MCAo, IGF-1 treatment significantly reduced brain uptake of Evan’s blue dye, while infarct volume was no different from controls. At 24h post MCAo, IGF-1 reduced infarct volume by 40% and decreased motor impairment (neurological score; p&lt;0.05). Lectin stained microvessels in the ischemic hemisphere displayed wider lumens (normal uncompressed lumen, p&lt;0.003) and more intense label (p&lt;0.01) in IGF-1 treated animals as compared to either vehicle or IGF-1+JB-1 groups. Ex vivo, IGF-1 reduced the loss of cell-cell contact due to OGD and reduced the dye transfer across the monolayer. Conclusions: Our findings indicate that IGF-1 acts in the early acute phase of ischemic stroke to reduce barrier permeability, which precedes its beneficial effect on infarct volume and behavior. Further, brain endothelial cells play a crucial role in IGF-1 mediated neuroprotection in middle-aged females.

Abstract WP96: The Histone Deacetylase Inhibitor, Sodium Butyrate, Exhibits Biphasic Neuroprotective Mechanisms in the Acute Phase of Ischemic Stroke in Middle-Aged Female Rats

Introduction: Sodium butyrate (NaB) is a histone deacetylase (HDAC) inhibitor exhibiting anti-inflammatory and neuroprotective effects in a rat ischemic model of a myocardial ischemia as well as stroke. Although clinical evidence shows that older women are at higher risk for stroke occurrence and greater stroke severity, no studies have evaluated the effectiveness of NaB either in females or in older animals. Methods: To determine the effects of NaB on stroke in older females, acyclic middle-aged Sprague-Dawley female rats (10-12 months old, constant diestrus) were subject to middle cerebral artery occlusion (MCAo) by intracerebral injection of recombinant endothelin-1. Rats were treated with NaB (300 mg/kg, i.p.) at 6h and 30h following ET-1 injection. Animals were tested for sensory motor performance pre and post stroke. Subsequently, rats were sacrificed at the early (2d) or late (5d) acute phase after MCAo. Serum and tissue samples were collected for biochemical analyses. Results: NaB treatment reduced infarct volume and ameliorated stroke-induced sensory motor impairment in middle-aged female rats post MCAo. At the early acute phase, NaB treatment decreased brain lipid peroxides, and reduced serum levels of GFAP, a marker of blood brain barrier permeability. NaB also reduced expression of the inflammatory cytokine IL-1beta in circulation and IL-18 in the ischemic hemisphere. At the late acute phase, NaB treatment further suppressed MCAo-induced increase of IL-1beta, IL-17A, and IL-18 in brain lysates (cortex and striatum) from the ischemic hemisphere, and decreased ischemia-induced upregulation of IL-1beta and IL-18 in circulation, indicating a potent anti-inflammatory effect of the HDAC inhibitor. Moreover, NaB treatment also increased expression of IGF-1, a known neuroprotectant, in peripheral tissues including serum, liver, and spleen. Conclusions: These data provide the first evidence that delayed (&gt; 6h) NaB treatment post-stroke is neuroprotective in older female rats. Importantly, these data also show that in addition to its well-known anti-inflammatory actions, NaB may exert a biphasic effect after stroke, operating initially to reduce oxidative stress in the brain, and later, elevating IGF-1 expression in peripheral tissues.

Abstract WP108: Astrocyte-specific rAAV-mediated Insulin-like Growth Factor-1 Expression in Aging Female Rats Alters Stroke-induced Brain Immune Profile and Reduces Motor Impairment

Introduction: Middle-aged female rats sustain larger strokes than younger female rats. This may be due to age-related loss of circulating and parenchymal brain levels of insulin like growth factor-1 (IGF-1). With age, IGF-1 synthesis is decreased in astrocytes, a critical cell type for post-stroke recovery. Here we tested the hypothesis that replenishing IGF-1 in aging astrocytes will improve stroke outcomes by influencing the type and extent of immune cells infiltration into the brain post-stroke. Methods: Middle-aged (10-12 mo old, acyclic) female rats were injected into the striatum and cortex with adeno-associated virus serotype 5 (rAAV5) packaged with the coding sequence of the hIGF-1 gene downstream of an astrocyte-specific promoter (GFAP). The rAAV-control consisted of an identical shuttle vector construct without the hIGF-1 gene. Three to four weeks later, animals underwent 90 minute transient middle cerebral artery occlusion via intraluminal suture. At 2d post stroke, flow cytometry was used to determine the type and extent of peripheral immune cells trafficked into the brain. In parallel studies, animals were tested for performance on sensory motor tasks at 2d and 5 days after MCAo. Results: rAAV-mediated IGF-1 expression was confirmed in astrocytes with RT-PCR. Flow cytometry analysis of immune cells in the brain at 24h post-stroke found that proportion of Treg cells was greater in animals with rAAV-IGF-1 as compared to rAAV-controls. Additionally, while there was no difference in the proportion of M2 microglia, rAAV-IGF-1 enhanced M2 infiltrating macrophages. At 2d and 5d post stroke, stroke-induced sensory motor impairment was reduced in animals with rAAV-IGF-1 as compared to rAAV-controls. Conclusion: Targeted enhancement of IGF-1 in astrocytes of middle-aged females improved stroke-induced behavioral impairment concomitant with recruitment of anti-inflammatory cell types to the ischemic brain.

Abstract WP102: Retrograde Neurodegeneration of Substantia Nigra Projections to the Striatum Following Long Term Survival After Ischemic Stroke

Introduction: Stroke survivors suffer from long-term physical, cognitive and affective disabilities. These disabilities lower the quality of life, contribute to social isolation and anhedonia, and clinical disorders such as post-stroke depression (PSD), which disproportionately affects women. Preclinical studies show that lesions of the substantia nigra (pars compacta) impair both motor performance and reward seeking behavior. Neurons from the SNc project to the striatum, a region that is significantly infarcted by middle cerebral artery occlusion (MCAo). Hypothesis: Ischemia induced by MCAo leads to retrograde degeneration of the SNc pathway projecting to the striatum. Methods: Middle-aged Sprague-Dawley female rats (12 months old) were subjected to ischemic stroke using a silicon-coated nylon filament suture to occlude the middle cerebral artery (MCA) which was removed 75min after to allow for reperfusion. After 14 weeks of survival, rats were again anesthetized and Fluorogold (Flg) was injected into the left and right striatum. Four days later, animals were overdosed with anesthetic, perfused with saline and formaldehyde and the brain removed for cryosectioning. In three sections per animal, Flg-labeled cells in the SNc were counted on both hemispheres, using fluorescent illumination. Results: Flg-injections into the striatum retrogradely labeled neurons in the midbrain. There was a 35% decrease (p&lt;0.009) in the number of Flg labeled SNc cells in the ischemic hemisphere (85.5+18.2) as compared to the non-ischemic hemisphere (132.4+22.7). The reduced number of projection neurons in the ischemic hemisphere is consistent with the loss of trophic support from the striatum. Conclusions: SNc neurons are a critical component of reward pathway and motor function, hence degeneration of these neurons could lead to long term motor deficits and depressive symptoms including anhedonia that are common after stroke.

Abstract 59: Post-stroke Delivery of Microrna-363 to Middle-aged Female Rats is Internalized by Neurons and Suppresses the Cell Death Effector Pathway

Background: MicroRNAs serve as translational inhibitors and offer a unique therapeutic target for acute diseases such as stroke. Profiling of circulating miRNA after stroke identified mir363, whose expression was inversely correlated with infarct volume. Middle aged female rats show worse stroke outcomes than younger females and have much lower levels of mir363. Our recent studies showed that iv injections of miR363 mimic to middle aged females, significantly improved stroke outcome. The present study is designed to determine the mechanisms by which miR-363 acts as a therapeutic miR. Methods: Middle aged (12 mo) females were subject to MCAo. At 4h post-stroke, animals received a tail-vein injection of miR-363-3p FAM or scrambled control. Animals were terminated at 48h or 5d post-MCAo and perfused transcardially or processed for protein, respectively. To determine which neural cell types localized exogenous mir363-3p, combined immunofluorescence was performed for cell specific markers (neuronal (NeuN), astrocytic (GFAP), microglial (CD11b) and endothelial (PECAM)) and mir363-3p-FAM mimic on coronal brain sections (25 mm thickness). Protein lysates from the ischemic tissue was analyzed for caspase-3 expression by Western blot analysis. Results: FAM-labeled mir363-3p was widely detected in the forebrain. The majority of NeuN+ cells in the cortex and striatum were also labeled with FAM-363-3p, indicating a robust internalization of the mimic in neurons. FAM-mir363-3p was also localized to a few microglia (CD11b +), virtually no double-label was seen in astrocytes and endothelial cells. Mir363 decreased the expression and functional activity of caspase3 in the ischemic hemisphere. Conclusion: Collectively, the data suggests that exogenous miR-363-3p is shuttled to the brain and is preferentially internalized by neurons. Together with the caspase-3 regulation, our data suggests that mir363 may improve stroke outcomes by suppressing a cell death effector.

Stereological quantification of age-related changes in myelinated fibers of rat white matter.

We investigated changes in myelin sheaths in the myelinated fibers of subcortical white matter in young (6-8 months old), middle-aged (18 months old), and elderly (27-28 months old) female Long-Evans rats using transmission electron microscopy and unbiased stereological techniques. We observed three age-related changes in myelin sheaths in the white matter of elderly rats: local splitting of the major dense line, myelin ballooning, and the formation of abundant myelin sheaths. Stereological analysis showed that the total length of myelinated fibers in white matter was 115±12 km (mean±SD) in young rats, 135±21 km in middle-aged rats, and 62±11 km in elderly rats. Myelinated fibers with diameters less than 1.0 μm and myelin sheath thicknesses less than 0.1 μm were significantly shorter in middle-aged and elderly rats compared with young rats. Age-related changes in myelin sheaths were evident in aged white matter. The loss of myelinated fibers with both small diameters and thin myelin sheaths is a potentially important change in aged white matter. Our results suggest that the myelin sheath changes in aged white matter may have important implications for age-related cognitive impairments.

The histone deacetylase inhibitor, sodium butyrate, exhibits neuroprotective effects for ischemic stroke in middle-aged female rats

BackgroundSodium butyrate (NaB) is a histone deacetylase (HDAC) inhibitor exhibiting anti-inflammatory and neuroprotective effects in a rat ischemic model of stroke as well as a myocardial ischemia model. Although clinical evidence shows that older women are at higher risk for stroke occurrence and greater stroke severity, no studies have evaluated the effectiveness of NaB either in females or in older animals.MethodsTo determine the effects of NaB on stroke in older females, acyclic middle-aged Sprague-Dawley female rats (9–11 months old, constant diestrus) were subject to middle cerebral artery occlusion (MCAo) by intracerebral injection of recombinant endothelin-1. Rats were treated with NaB (300 mg/kg, i.p.) at 6 and 30 h following ET-1 injection. Animals were sacrificed at the early (2 days) or late (5 days) acute phase after MCAo. Serum and tissue lysates were collected for biochemical analyses.ResultsNaB treatment reduced infarct volume and ameliorated sensory motor impairment in middle-aged female rats, when measured at 2 and 5 days post MCAo. At the early acute phase (2 days post stroke), NaB treatment decreased brain lipid peroxides, and reduced serum levels of GFAP, a surrogate marker of blood-brain barrier (BBB) permeability. NaB also reduced expression of the inflammatory cytokine IL-1beta in circulation and IL-18 in the ischemic hemisphere. At the late acute phase (5 days post stroke), NaB treatment further suppressed MCAo-induced increase of IL-1beta, IL-17A, and IL-18 in brain lysates (cortex and striatum) from the ischemic hemisphere, and decreased ischemia-induced upregulation of IL-1beta and IL-18 in circulation, indicating a potent anti-inflammatory effect of the HDAC inhibitor. Moreover, NaB treatment also increased expression of IGF-1, a known neuroprotectant, in peripheral tissue including serum, liver, and spleen at the late acute phase.ConclusionsThese data provide the first evidence that delayed (>6 h) NaB treatment post-stroke is neuroprotective in older female rats. Additionally, these data also show that in addition to its well-known anti-inflammatory actions, NaB may exert a biphasic effect after stroke, operating initially to reduce BBB permeability and oxidative stress in the brain, and later, elevating IGF-1 expression in peripheral tissues.

17β-estradiol replacement therapy protects myelin sheaths in the white matter of middle-aged female ovariectomized rats: a stereological study

Many studies have shown that estrogen replacement therapy (ERT) can improve cognitive function and affect the structure of the brain, including the white matter, in postmenopausal women. However, it is unclear whether ERT plays an important role in white matter remodeling in postmenopausal women. In the present study, middle-aged (9–12-month-old) female Sprague-Dawley rats were bilaterally ovariectomized (OVX) and randomly allocated to the vehicle treatment (OVX+Veh) group or the 17β-estradiol replacement (OVX+E) group. After 1 month of treatment, spatial learning and memory capacities were assessed using the Morris water maze task. Then, stereological methods were used to quantitatively evaluate white matter volume and myelinated fiber parameters of the white matter in the 2 groups of rats. The results revealed that the mean escape latency of the OVX+E rats in the Morris water maze task was significantly shorter than that of the OVX+Veh rats. The volume density of the myelinated fibers and the volume density and total volume of the myelin sheaths were significantly greater in the OVX+E rats than in the OVX+Veh rats. However, there were no significant differences in white matter volume or in the total length or volume of myelinated fibers in white matter between the 2 groups of rats. Our results showed that 1 month of ERT had significant beneficial effects on spatial learning capacity and on the myelin sheaths and myelinated fibers in the white matter of middle-aged OVX rats.

Cardioprotective effect of a new glutamic acid derivative, glufimet, under conditions of acute immobilization and painful stress in animals of different ages

The effect of 24-hour immobilization and painful stress on myocardial contractility was studied in young (6-month), middle-aged (12-month) and old (24-month) female rats. A reduction of the functional heart reserves was revealed. This manifested itself in a lower growth of the myocardial contraction and relaxation rates (+dP/dt max and–dP/dt max), the left ventricular pressure (LVP), the maximum intensity of the functioning of the structures (MIFS) under conditions of increased pre- and afterload, and stimulation of the cardiac adrenergic receptors, which was especially pronounced in the group of 24-month-old rats. During the load tests, higher growth indicators of +dP/dt max,–dP/dt max, LVP, and MIFS were observed in animals of all age groups treated with glufimet at a dose of 29 mg/kg before and after stress; these indicators were most significant in older rats compared to young and middle-aged animals. A reference drug, Phenibut, improves the studied parameters equally in 6-, 12-, and 24-month-old females subjected to stress.

The Role of RFamide-Related Peptide-3 in Age-Related Reproductive Decline in Female Rats.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic–pituitary–gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system.

Estrogen-induced neuroprotective and anti-inflammatory effects are dependent on the brain areas of middle-aged female rats.

Reproductive aging in females is characterized by fluctuations and precipitous decline in estrogen levels, which may lead to reduction in cognitive function and age-associated neurodegenerative disorders. The nature of estrogen-mediated neuronal plasticity is unknown during reproductive aging. We hypothesize that estrogen treatment of early middle-aged ovariectomized rats may exert specific effects in the brain by modulating signaling pathways regulating metabolic enzymes, inflammatory markers, antioxidant status, cholinergic function and survival signals. To investigate the mechanisms of estrogen-induced effects on neuroprotection and neuroinflammation through the involvement of intracellular signaling pathways in brain areas of ovariectomized (OVX) middle-aged (MA) female rats. Ovariectomized early MA female Sprague-Dawley rats (n=8/group) were implanted with 17β-estradiol (E2) 30-day release pellets (0.6μg and 300μg). At the end of the treatment period, frontal cortex (FC), striatum (STR), medial basal hypothalamus (MBH), and hippocampus (HP) were isolated and examined for the expression of tyrosine hydroxylase (p-TH), nerve growth factor (NGF), p-NF-κB (p50 and p65)and p-ERK, p-CREB, p-Akt, and activities of cholinesterases and antioxidant enzymes, key regulatory enzymes of metabolic pathways, and nitric oxide production. E2 enhanced p-TH expression in FC and HP, reduced NGF expression in HP, and suppressed p-NF-κB expression in FC and STR. It also increased the expression of molecular markers (p-ERK, p-CREB and p-Akt), and nitric oxide production in various brain areas, while differentially regulating the activities of metabolic enzymes and cholinesterases. Estrogen modulates the neural and inflammatory factors, and intracellular markers depending on the brain areas that may influence differential remodeling of neuronal circuitry which can be used to develop therapeutic strategies in cognitive impairment and neurodegenerative disorders in aging.

The Post-Ovariectomy Interval Affects the Antidepressant-Like Action of Citalopram Combined with Ethynyl-Estradiol in the Forced Swim Test in Middle Aged Rats.

The use of a combined therapy with low doses of estrogens plus antidepressants to treat depression associated to perimenopause could be advantageous. However the use of these combinations is controversial due to several factors, including the time of intervention in relation to menopause onset. This paper analyzes whether time post-OVX influences the antidepressant-like action of a combination of ethynyl-estradiol (EE2) and citalopram (CIT) in the forced swim test (FST). Middle-aged (15 months old) female Wistar rats were ovariectomized and after one or three weeks treated with EE2 (1.25, 2.5 or 5.0 µg/rat, s.c.; −48 h) or CIT (1.25, 2.5, 5.0 or 10 mg/kg, i.p./3 injections in 24 h) and tested in the FST. In a second experiment, after one or three weeks of OVX, rats received a combination of an ineffective dose of EE2 (1.25 µg/rat, s.c., −48 h) plus CIT (2.5 mg/kg, i.p./3 injections in 24 h) and subjected to the FST. Finally, the uteri were removed and weighted to obtain an index of the peripheral effects of EE2 administration. EE2 (2.5 or 5.0 µg/rat) reduced immobility after one but not three weeks of OVX. In contrast, no CIT dose reduced immobility at one or three weeks after OVX. When EE2 (1.25 µg/rat) was combined with CIT (2.5 mg/kg) an antidepressant-like effect was observed at one but not three weeks post-OVX. The weight of the uteri augmented when EE2 was administrated three weeks after OVX. The data suggest that the time post-OVX is a crucial factor that contributes to observe the antidepressant-like effect of EE2 alone or in combination with CIT.

Ovarian hormones, but not fluoxetine, impart resilience within a chronic unpredictable stress model in middle-aged female rats

Depression is more prevalent in women than in men, and women are at a heightened risk for depression during the postpartum and perimenopause. There is also evidence to suggest that the ovarian hormone milieu may dictate antidepressant efficacy. Thus, it is important to investigate the role of ovarian hormones in the pathogenesis of depression and in the mechanisms that may underlie antidepressant efficacy. In the present study, we used 10-month-old female Sprague-Dawley rats to examine the effects of long-term ovarian hormone deprivation on the development of depressive-like endophenotypes after chronic stress, and on antidepressant efficacy. Four months following ovariectomy (OVX) or sham surgery, all rats were subjected to 6 weeks of chronic unpredictable stress (CUS). During the last 3 weeks of CUS, rats received daily injections of fluoxetine (5 mg/kg) or vehicle. All rats were assessed on measures of anxiety- and depressive-like behavior, hypothalamic-pituitary-adrenal (HPA) negative feedback inhibition, and on markers of neurogenesis and microglia in the dentate gyrus. Our findings demonstrate that long-term ovarian hormone deprivation increased anxiety and depressive-like behavior, as seen by increased immobility in the forced swim test and latency to feed in the novelty suppressed feeding test, and decreased sucrose preference. Further, long-term OVX resulted in impaired HPA negative feedback inhibition, as seen in the dexamethasone suppression test. Fluoxetine treatment showed limited behavioral and neuroendocrine efficacy, however it reduced microglial (Iba-1) expression, and increased cell proliferation, neurogenesis (via cell survival), and the expression of the polysialylated neuronal cell adhesion molecule (PSA-NCAM) in the dentate gyrus, although these effects varied by region (dorsal, ventral) and ovarian status. Taken together, our findings demonstrate that ovarian hormones may impart resilience against the behavioral and neuroendocrine consequences of chronic unpredictable stress, and may modulate the effects of fluoxetine on cell proliferation, neurogenesis, and PSA-NCAM in the middle-aged female.