Hippocampus Of Middle-aged Rats Research Articles

Effects of Age and Tocotrienol-Rich Fraction on Mitochondrial Respiratory Complexes in the Hippocampus of Rats

Mitochondrial dysfunction is common in the brain with age. Prevention of mitochondrial dysfunction at an early age may protect the brain against neurodegeneration in later life. Tocotrienol-rich fraction (TRF) has been reported to be neuroprotective in old rats, but its effect remains unknown for middle-aged animals. This study aimed to determine the effect of TRF on activities of mitochondrial respiratory chain complexes in the hippocampus of middle-aged rats. Male Sprague Dawley rats were divided into 4 groups: young control (3 months old), adult control (12 months old), adult rats supplemented with palm kernel oil (PKO) as the vehicle, and adult rats supplemented with TRF by gavage at 200 mg/kg body weight/day for 3 months. At the end of the supplementation, activities of complex I, I+III, II, II+III, III, IV, and citrate synthase in the isolated mitochondria of the hippocampus were measured by spectrophotometry. Complex II activity was higher, while citrate synthase activity was lower in adult rats than in young rats. A decrease of citrate synthase activity suggests loss of mitochondrial mass and intactness in the hippocampus at middle age. Interestingly, PKO-treated adult rats had lower complex I and IV activities, but higher complex I+III activity than adult control rats. These findings indicate PKO modulated activities of the complexes. In TRF-treated adult rats, the complex I activity was higher, while the complex IV activity was lower than PKO-treated adult rats. TRF restored the complex I activity and may have the potential to reverse complex I deficiency.

Proteomic Profile of Carbonylated Proteins Screen the Regulation of Calmodulin-Dependent Protein Kinases-AMPK-Beclin1 in Aerobic Exercise-Induced Autophagy in Middle-Aged Rat Hippocampus

Background: Carbonylation is an oxidative modification of the proteins and a marker of oxidative stress. The accumulation of toxic carbonylated proteins might be one of the onsets of pathogenesis in hippocampal aging or neurodegeneration. Enormous evidence indicates that regular aerobic exercise might alleviate the dysfunction of carbonylated proteins, but the adaptational mechanisms in response to exercise are unclear. Objective: This study explored the carbonyl stress mechanism in the hippocampus using proteomics and the role of calmodulin-dependent protein kinases (CAMK)-AMP-activated protein kinase (AMPK)-Beclin1 signaling pathways in alleviating aging or improving function with regular aerobic exercise. Methods: Twenty-four healthy 13-month-old male Sprague-Dawley rats (average 693.21 ± 68.85 g) were randomly divided into middle-aged sedentary control group (M-SED, n = 12) and middle-aged aerobic exercise runner group (M-EX, n = 12). The M-EX group participated in regular aerobic exercise – treadmill running – with exercise intensity increasing gradually from 50–55% to 65–70% of maximum oxygen consumption (V˙O2max) over 10 weeks. The targeted proteins of oxidative modification were profiled by avidin magnetic beads and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Western blots were used to test for molecular targets. Results: Regular aerobic exercise restores the intersessional habituation and rescues the hippocampus morphological structure in middle-aged rats. ­ESI-Q-TOF-MS screened 56 carbonylated proteins only found in M-SED and 16 carbonylated proteins only found in M-EX, indicating aerobic exercise decreased carbonyl stress. Intriguingly, Ca²⁺/CAMK II alpha (CAMKIIα) was carbonylated only in the M-SED group at the oxidative modification site of 4-hydroxynonenal adducts, while regular aerobic exercise alleviated CAMKIIα carbonylation. Regular aerobic exercise significantly increased the expression and phosphorylated, active levels of CAMKIIα and AMPKα1. It also upregulated the expression of Beclin1 and microtubule-associated protein1-light chain 3 in the hippocampus. Conclusion: Quantification of CAMKIIα carbonylation may be a potential biomarker of the hippocampal senescence. Additionally, regular aerobic exercise-induced autophagy via the activation of CAMK-AMPK-Beclin1 signaling pathway may mitigate the hippocampal neurodegeneration or pathological changes by alleviating protein carbonylation (carbonyl stress).

Effect of Initial Aging and High-Fat/High-Fructose Diet on Mitochondrial Bioenergetics and Oxidative Status in Rat Brain.

Middle age is an early stage of the aging process, during which the consumption of diets rich in saturated fats and/or simple sugars might influence brain function, but only few data are available on this issue. We therefore investigated the impact of a diet rich in saturated fat and fructose (HFF) on mitochondrial physiology in hippocampus and frontal cortex of middle-aged rats (1year old), by including a group of adult rats (90days) as a "negative control," lacking the putative effect of aging. Middle-aged rats were fed HFF or control diet for 4weeks. Mitochondrial function was analyzed by high-resolution respirometry and by assessing the amount of respiratory complexes. Markers of oxidative balance, as well as the protein content of uncoupling protein 2 (UCP2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and peroxisome proliferator-activated receptor alpha (PPARα), were also assessed. A decrease in the activity of complex I was detected in both brain areas of middle-aged rats. In hippocampus, mitochondrial respiratory capacity and complex IV content decreased with age and increased with HFF diet. Higher protein oxidative damage, decreased antioxidant defenses, and increased UCP2 and PGC-1α content were found in hippocampus of middle-aged rats. HFF feeding induced a significant reduction in the amount of UCP2, PGC-1α, and PPARα, together with higher protein oxidative damage, in both brain areas. Overall, our results point to middle age as a condition of early brain aging for mitochondrial function, with hippocampus being an area more susceptible to metabolic impairment than frontal cortex.

Effect of β-alanine supplementation on carnosine and histidine content in the hippocampus of 14-month-old rats.

Carnosine and histidine content in the hippocampus of 14-month-old male rats was examined following 30 days of β-alanine supplementation. All animals were provided identical diets; however, 100 mg of β-alanine was mixed with glucomannan (80:20 blend) in the water of β-alanine supplemented animals. Hippocampal carnosine content was significantly greater (p = 0.005) for β-alanine compared with control, while no differences (p = 0.438) were noted in histidine content between groups. Results provide initial evidence that β-alanine supplementation increases carnosine content in the hippocampus of middle-aged rats, without compromising histidine content. β-Alanine supplementation increases hippocampal carnosine content without compromising histidine content in middle-aged rats.

Evaluation of Rosmarinus officinalis L. extract on spatial memory, learning and antioxidant enzymes activities in the hippocampus of rats

The Rosemarinus officinalis extract (RE) possesses various antioxidant, cytoprotective and cognition- improving bioactivities. In this study, we postulated which doses of RE have a more effect on the hippocampus of middle-aged rats. In this experimental study, thirty-two middle-aged swiss albino rats were fed by different doses (50, 100 and 200 mg/kg/day) of RE (containing 40% carnosic acid) or distilled water for 12 weeks. The effects of different RE doses on learning and spatial memory scores, hippocampal neuronal survival, antioxidant enzymes and lipid peroxidation amount were evaluated by one and two way analysis of variance (ANOVA). It seemed that RE (100mg/kg) could recover the spatial memory retrieval score (p< 0.05). The amount of activity of SOD, GPx and CAT enzymes in the hippocampus of animals of the RE (100mg/kg) group showed a significant increase compared to the normal group (p< 0.01), (p< 0.01) and (p< 0.05), respectively. Also, the amount of activity of GPx in the RE (100 mg/kg) group of animals showed a significant increase compared to the normal group (p< 0.05). No significant difference was found between the groups in the MDA level. The results revealed that RE (40% carnosic acid) may improve the memory score and oxidative stress activity in middle aged rats in a dose dependent manner, especially in 100mg/kg.

Impaired insulin signaling and spatial learning in middle-aged rats: The role of PTP1B

The insulin and Brain-Derived Neurotrophic Factor (BDNF) signaling in the hippocampus promotes synaptic plasticity and memory formation. On the other hand, aging is related to the cognitive decline and is the main risk factor for Alzheimer's Disease (AD). The Protein-Tyrosine Phosphatase 1B (PTP1B) is related to several deleterious processes in neurons and emerges as a promising target for new therapies. In this context, our study aims to investigate the age-related changes in PTP1B content, insulin signaling, β-amyloid content, and Tau phosphorylation in the hippocampus of middle-aged rats. Young (3 months) and middle-aged (17 months) Wistar rats were submitted to Morris-water maze (MWM) test, insulin tolerance test, and molecular analysis in the hippocampus. Aging resulted in increased body weight, and insulin resistance and decreases learning process in MWM. Interestingly, the middle-aged rats have higher levels of PTP-1B, lower phosphorylation of IRS-1, Akt, GSK3β, mTOR, and TrkB. Also, the aging process increased Tau phosphorylation and β-amyloid content in the hippocampus region. In summary, this study provides new evidence that aging-related PTP1B increasing, contributing to insulin resistance and the onset of the AD.

EPA/DHA and Vitamin A Supplementation Improves Spatial Memory and Alleviates the Age-related Decrease in Hippocampal RXRγ and Kinase Expression in Rats.

Studies suggest that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and vitamin A are critical to delay aged-related cognitive decline. These nutrients regulate gene expression in the brain by binding to nuclear receptors such as the retinoid X receptors (RXRs) and the retinoic acid receptors (RARs). Moreover, EPA/DHA and retinoids activate notably kinase signaling pathways such as AKT or MAPK, which includes ERK1/2. This suggests that these nutrients may modulate brain function in a similar way. Therefore, we investigated in middle-aged rats the behavioral and molecular effects of supplementations with EPA/DHA and vitamin A alone or combined. 18-month-old rats exhibited reference and working memory deficits in the Morris water maze, associated with a decrease in serum vitamin A and hippocampal EPA/DHA contents. RARα, RXRβ, and RXRγ mRNA expression and CAMKII, AKT, ERK1/2 expression were decreased in the hippocampus of middle-aged rats. A combined EPA/DHA and vitamin A supplementation had a beneficial additive effect on reference memory but not in working memory in middle-aged rats, associated with an alleviation of the age-related decrease in RXRγ, CAMKII, AKT, and ERK1 expression in the hippocampus. This study provides a new combined nutritional strategy to delay brain aging.

Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1month old) and middle-aged (7-12month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.

Middle-aged rat hippocampus and some early changes accompanying aging

Abstract

Effects of a 4 month enriched environment on the hippocampus and the myelinated fibers in the hippocampus of middle-aged rats

An enriched environment has been shown to enhance learning and memory and to induce morphological changes in the hippocampus. In the present study, 14-month (middle-aged) female and male Sprague–Dawley rats were randomly divided into enriched environment (EE) rats and standard environment (SE) rats. EE rats were reared in an enriched environment and SE rats were reared in a standard environment for 4months. The spatial learning capacity was assessed with Morris water maze. The hippocampus and the myelinated fibers in the rat hippocampus were quantitatively investigated with a transmission electronic microscope technique and stereological methods. The female rats housed in an enriched environment showed improved performance in the Morris water maze. There was no significant difference in the total volume of hippocampus between SE rats and EE rats. The total length and total volume of the myelinated fibers in the hippocampus of the female and male EE rats were significantly increased, respectively, when compared to the female and male SE rats. The increase of the total length of the myelinated nerve fibers in the hippocampus was mainly due to the increase of the myelinated fibers with diameters from 0.5 to 0.9μm. Our results showed that a 4month enriched environment had significant effects on the spatial learning capacity and the myelinated fibers in the hippocampus of middle-aged rats. The present study might provide an important theoretical basis for searching for an ethological strategy to delay the progress of brain aging in the future.

Changes in tyrosine‐nitrated proteins in the hippocampus of adult‐and middle‐aged rat

Nitration of tyrosine residues in proteins is widely used as a marker for oxidative or nitrative stress in animal cells. Increase in tyrosine‐nitrated proteins with age in rat hippocampus and identification of several tyrosine‐nitrated proteins from aged rat muscles have been reported. However, no study on the protein nitration in the brain of adult‐ and middle‐aged rats has been demonstrated yet. In this study, we determined relative contents of nitrated proteins, nitric oxide synthases (NOS), and superoxide dismutases (SOD) in the hippocampus of adult‐ and middle‐aged F344 rats. We also determined difference in nitrated proteins between the adult‐ and middle‐aged rat hippocampus by using two‐dimensional electrophoresis (2DE). Western blot analyses showed that relative contents of nitrotyrosine‐containing proteins and expression of Mn‐SOD, EC‐SOD, nNOS, and eNOS were not different between the adult‐ and middle‐aged rats. However, the expression of Cu, Zn‐SOD was increased in the middle‐aged rat compared with that of adult rat. Furthermore, several spots for tyrosine‐nitrated protein in 2DE were different between the adult‐ and middle‐aged rats. Thus, although the nitrative and oxidative stress may not different so much between the adult‐ and middle‐aged rat hippocampus, target proteins for the nitration are changed. Proteome analysis of the nitrated proteins is underway in our laboratory.

Diet-induced effects on neuronal and glial elements in the middle-aged rat hippocampus

Consumption of a high-fat and/or high-cholesterol diet can have detrimental effects on the brain. In the present study, dietary treatment with saturated fats, trans fats, or cholesterol to middle-aged Fischer 344 rats resulted in alterations to serum triglyceride and cholesterol levels, organ weights, and hippocampal morphology. Previously, we demonstrated that a 10% hydrogenated coconut oil and 2% cholesterol diet resulted in worse performance on the 12-day water radial arm maze, increased cholesterol and triglyceride levels, and decreased dendritic microtubule associated protein 2 (MAP2) staining in the hippocampus. The diets administered herein were used to examine components from the previous diet and further examine their effects on hippocampal morphology. Specifically, neuronal morphology, dendritic integrity, fatty acid metabolism, microgliosis, and blood vessel structure in the hippocampus and/or adjacent structures were explored. Our results indicate alterations to peripheral and neural systems following each of the diets.