Chronic stress attenuates glucocorticoid negative feedback: involvement of the prefrontal cortex and hippocampus

Disruption of the glucocorticoid negative feedback system is observed in approximate one half of human depressives, and a similar condition is induced in animals by chronic stress. This disruption is thought to involve down-regulation of glucocorticoid receptors (GRs) in the feedback sites of the brain. However, the responsible site of the brain has not been well elucidated. Here we examined the effects of chronic stress induced by water immersion and restraint (2 h/day) for 4 weeks followed by recovery for 10 days on the GR levels in the prefrontal cortex (PFC), hippocampus, and hypothalamus of rats using a Western immunoblot technique. In the PFC, the cytosolic GR levels were decreased, but the nuclear GR levels were not changed. In the hippocampus, the levels of cytosolic and nuclear GRs were increased. However, there were no marked changes in the GR levels in the hypothalamus. The changes in the cytosolic GR levels were confirmed at the mRNA level by an in situ hybridization technique. We next examined the suppressive effects of dexamethasone (DEX) infusions into these regions on the circulating corticosterone levels. When DEX was infused into the PFC or hippocampus of the chronically stressed rats, the suppressive response to DEX was abolished, but the response was normal in the hypothalamus. In addition, when DEX was injected systemically to the chronically stressed rats, the suppressive response to DEX was significantly attenuated. These results suggest that the abnormal changes in GRs in the higher centers of the hypothalamo-pituitary-adrenal axis are involved in the chronic stress-induced attenuation of the feedback. Since dysfunction of the PFC or hippocampus is implicated in the pathogenesis of depression, the present findings would help to understand the mechanisms underlying the disrupted feedback system and its relation to brain dysfunction in depression.     

Temporal and spatial dynamics of corticosteroid receptor down-regulation in rat brain following social defeat

The experiments explored the nature and time course of changes in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) binding in homogenates of various brain regions and pituitary of male Wistar rats following social defeat stress. One week after defeat, the binding capacity of GRs was decreased in the hippocampus and the hypothalamus while no changes were observed in the parietal cortex and the pituitary. The number of MRs remained at the same level as in undefeated rats. Three weeks postdefeat, the initially down-regulated GR returned to baseline level in the hippocampus and the hypothalamus. However, GR binding was now decreased in the parietal cortex. Severe down-regulation of MRs was detected in the hippocampal and septal tissue. The results show that brief but intense stress like social defeat induces a long-lasting down-regulation of corticosteroid receptors and that the temporal dynamics of these changes are not only differential for GRs and MRs but also for brain sites.     

Aging increases basal but not stress-induced levels of corticosterone in the brain of the awake rat

The main purpose of this study was to evaluate the effect of aging on plasma and free corticosterone (CORT) levels in the brain in basal conditions and in response to an acute stressor. Microdialysis experiments were performed in the hippocampus (HC) and the prefrontal cortex (PFC) of young adult (6 months) and aged (24 months) male Wistar rats. Basal free levels of CORT in the HC and the PFC were higher in aged animals. Restraint stress increased plasma CORT and free CORT levels in the HC and the PFC both in young and aged animals. However, while the increase of plasma CORT was higher in aged rats compared with young rats, the increases of free CORT in the HC and the PFC were not different between these two groups of rats. These results suggest that the changes produced by aging in the brain may be related to the enhanced basal levels of free CORT and not to the CORT increases in response to stress.     

Age-related spatial working memory impairment is caused by prefrontal cortical dopaminergic dysfunction in rats

There is evidence of prefrontal cortex (PFC)–dependent cognitive deficits, such as working memory impairment, during the normal aging process in humans and animals. Although working memory function and the PFC dopaminergic system are thought to be closely related, the relationship between them in aged subjects remains unclear. The present study was aimed to clarify the involvement of PFC dopaminergic activity in age-related working memory impairment. For this purpose, we examined working memory in young (3-month-old) and aged (24-month-old) rats, using the T-maze delayed alternation task. As a result, delayed alternation performance was impaired in aged rats compared to young rats, indicating age-related working memory impairment. In addition, aged rats showed reduced dopaminergic transmission in the prelimbic cortical region of the PFC, concomitant with attenuated tyrosine hydroxylase activity in the PFC, but not in the ventral tegmental area and substantia nigra, which was evaluated immunohistochemically and enzymatically. Moreover, age-related working memory impairment was improved by direct stimulation of the prelimbic cortical region of the PFC with 10 or 30 ng, but not 100 ng, of a D1 receptor agonist, SKF 81297, indicating that the SKF 81297 response was an inverted “U” pattern. The maximum SKF 81297 response (30 ng) was abolished by a D1 receptor antagonist, SCH 23390. Thus, age-related working memory impairment was through reduced PFC dopaminergic transmission caused by decreased dopamine synthesis in the prefrontal termination region, but not at the site where the projections originate. This finding provides direct evidence showing the involvement of dopaminergic dysfunction in the development of PFC cognitive deficits during the normal aging process and would help to understand the aging physiology and pathology of the brain.     

Influence of aging on chondroitin sulfate proteoglycan expression and neural stem/progenitor cells in rat brain and improving effects of a herbal medicine, yokukansan

There is evidence of structural and functional deterioration in the brain, including the prefrontal cortex (PFC) and hippocampus, during the normal aging process in animals and humans. Extracellular matrix-associated glycoproteins, such as chondroitin sulfate proteoglycans (CSPGs), are involved in not only maintaining the structures and functions of adult neurons, but also regulating the proliferation, migration, and neurite outgrowth of neural stem cells in the brain. On the other hand, a herbal medicine, yokukansan (YKS), is used in a variety of clinical situations for treating symptoms associated with age-related neurodegenerative disorders such as Alzheimer's disease, but its pharmacological properties have not been fully understood. The present study was designed to clarify the influence of aging and the improving effects of YKS on the expression of aggrecan, a major molecule of CSPGs, and on the proliferation and migration of neural stem/progenitor cells identified by bromodeoxyuridine (BrdU) incorporation in the PFC and hippocampus including the dentate gyrus. Aged rats (24 months old) showed a significant increase in aggrecan expression throughout the PFC and in the hippocampus particularly in the CA3 subfield, but not the dentate gyrus compared to young rats (5 months old), evaluated by the immunohistochemical method. YKS treatment decreased the age-related increase in aggrecan expression as well as normal expression in young rats. Aged rats also showed a decreased number of BrdU-labeled cells in the PFC and hippocampus, and these decreases were improved by YKS treatment, which also increased the numbers in young rats. These results suggest that aging influences the microenvironment for adult and immature neurons in the brain, which may affect the proliferation and migration of neural stem/progenitor cells, and YKS has pharmacological potency for these age-related events. These findings help to understand the physiology and pathology of the aged brain and provide an anti-aging strategy for the brain.     

Oxytocin concentration changes in different rat brain areas but not in plasma during aging

The concentration of oxytocin was measured by radioimmunoassay in different brain areas, hypothesis, and plasma of male Wistar Kyoto rats during aging. Although no difference in the concentration of oxytocin in any of the above tissues among 2- and 6-month-old rats was found, in 12-month-old rats a 21 % decrease was observed in both septum and hippocampus, but not in the hypothalamus, hypophysis, and plasma, when compared to values of 2- and 6-month-old rats. In 18-month-old rats, the decrease of septal and hippocampal oxytocin content was higher than that found in 12-month-old rats, but no change was found in the hypothalamus, neurohypophysis, and plasma. In 24-month-old rats, oxytocin content was similar to that found in 18-month-old rats in all tissues analyzed. The results suggest that aging induces an impairment of oxytocinergic transmission in the central nervous system but not in the neurohypophyseal system.     

Regional changes in the metabolite profile after long-term hypoxia-ischemia in brains of young and aged rats: a quantitative proton MRS study

Quantitative proton magnetic resonance spectroscopy (MRS) was used to determine region-specific metabolic changes in young and aged animals subjected to a long-term hypoxic-ischemic injury. Focal ischemia, which was studied as an experimental stroke model, was induced in 3- and 24-month-old rats by unilateral common carotid artery occlusion associated with 24 h of hypoxia. Eight metabolites were quantified from extracts in three different brain regions (hippocampus, frontoparietal and occipital cortices) from both the ipsilateral and contralateral sides. Our findings showed significant differences in lactate and myo-inositol concentration values in the hippocampus of the aged rats as compared to the same area of the young adult group under normoxic conditions. After hypoxia-ischemia (HI), the most relevant changes in metabolite concentrations were found in the hippocampal region of both young and aged groups as compared to their age-matched controls. Of the three brain areas under investigation, the hippocampus proved to be particularly susceptible to the prolonged hypoxia-ischemia perturbation. The effects were more evident in the aged animals.     

Effect of tibolone administration on central and peripheral levels of allopregnanolone and beta-endorphin in female rats

OBJECTIVE: We evaluated the effects of tibolone oral administration on neuroendocrine function by investigating the modulation exerted by tibolone administration on allopregnanolone and central and peripheral beta-endorphin (beta-EP) levels in ovariectomized rats. DESIGN: Female Wistar rats (N = 64) were included: 48 rats were ovariectomized, 8 cycling rats were included as controls, and 8 cycling rats were treated with placebo. The ovariectomized animals were divided into six groups: untreated rats and those that received 14-day oral treatment with either placebo, estradiol valerate (E2V) 0.05 mg/kg/d, or tibolone (0.1, 0.5, or 2 mg/kg/d. beta-EP levels were assessed in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, neurointermediate pituitary, and plasma, whereas allopregnanolone levels were measured in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, adrenal glands, and serum. RESULTS: The administration of tibolone (0.5 and 2 mg/kg/d) in ovariectomized rats induces a significant increase of allopregnanolone in the frontal lobe, parietal lobe, hippocampus, hypothalamus, whereas in serum a significant increase of allopregnanolone occurs only with the dose of 2 mg/kg/d, a significant decrease in allopregnanolone levels occurs in the adrenal glands. No changes occurred in the anterior pituitary. Tibolone doses of 0.5 and 2 mg/kg/d induced a significant increase in beta-EP content in the frontal lobe, hypothalamus, and neurointermediate lobe; and, at doses of 2 mg/kg/d, in the parietal lobe, anterior pituitary, and plasma, without changes in the hippocampus. Compared with E2V, 0.5 mg/kg/d tibolone showed a similar effect on allopregnanolone and beta-EP in most brain regions. CONCLUSIONS: Tibolone administration affects beta-EP and allopregnanolone levels, playing a role as a neuroendocrine modulator.     

Effect of aging and acetyl-L-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl-L-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl-L-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.     

Aging of the serotonergic system in the rat forebrain: an immunocytochemical and neurochemical study.

Age-related changes in both morphological and neurochemical parameters of indol- and catecholaminergic system in the rat brain were examined. A qualitative histochemical survey of the occurrence of aberrant serotonergic fibers in the aged rat brain suggests region-specificity in the process of degeneration. Forebrain areas, such as the caudate-putamen complex, globus pallidus, prefrontal and frontoparietal cortices were consistently affected, whereas serotonergic fibers were only infrequently affected in other areas like septal and amygdaloid nuclei. Neurochemical data similarly revealed regional differences. 5-Hydroxytryptamine levels were increased in the frontoparietal cortex, hippocampus, hypothalamus and the mesencephalic raphe region but remained unchanged in the caudate-putamen complex. 5-Hydroxyindolacetic acid levels were also enhanced in all these areas. Examination of brains of 12-, 18- and 24-month-old rats revealed that aberrant serotonergic fibers were already present at the age of 12 months and their incidence increase with age. There was no difference in the number of serotonergic cells in the dorsal raphe nucleus of young and aged rats. Aberrant tyrosine hydroxylase-immunoreactive fibers were observed only infrequently. Their occurrence showed no overlap with the areas containing aberrant serotonergic fibers. Neurochemical estimates of the levels of catecholamines in young versus aged rat brain areas similarly revealed regional and neurotransmitter specific differences to occur during the process of aging.     

Muscarinic receptor-mediated GTP-Eu binding in the hippocampus and prefrontal cortex is correlated with spatial memory impairment in aged rats

The present study examined muscarinic receptor/G-protein coupling in the hippocampus and the prefrontal cortex of young and aged Long-Evans rats characterized for spatial learning ability in the Morris water maze. In a highly sensitive time-resolved fluorometry GTP-Eu binding assay, muscarinic-mediated GTP-Eu binding was severely blunted in hippocampus (-32%) and prefrontal cortex (-34%) as a consequence of aging. Furthermore, the magnitude of decreased muscarinic-mediated GTP-Eu binding was significantly correlated with the severity of spatial learning impairment in hippocampus and prefrontal cortex of aged rats and was specifically decreased in the subset of aged rats that were spatial learning impaired when compared to the aged unimpaired and the young rats. Western blot data indicated a preservation of the membrane-bound M1 receptor and the Galphaq/11 protein in both brain regions. These data demonstrate that muscarinic signaling is severely impaired as a consequence of normal aging in a manner that is closely associated with age-related cognitive decline.     

Aging but not estrogen alters regional [3H]5-HT binding in the CNS of female Fischer 344 rats

Age-related changes in serotonergic regulation of neuroendocrine function were investigated in female Fischer 344 rats; serotonin ([3H]5-HT) binding sites were characterized in several brain regions. Neither the number (Bmax) nor the affinity (Kd) of [3H]5-HT sites were altered in the frontal cortex of reproductively young and senescent groups. However, a significant decline in receptor affinity was observed in the hypothalamus and midbrain dorsal raphe nucleus. An increase in the density of binding sites was also observed in the hypothalamus with advancing age. Acute 48 h exposure to estrogen failed to influence [3H]5-HT binding site characteristics in these brain regions. In summary, these results suggest that age-related changes in [3H]5-HT binding are regionally specific. Moreover, the observed changes in hypothalamic 5-HT function may underlie neuroendocrine aging events.     

Ladostigil prevents age-related glial activation and spatial memory deficits in rats

Oxidative stress and glial activation occur in the aging brain. Ladostigil is a new monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitor designed for the treatment of Alzheimer's disease. It has neuroprotective and antioxidant activities in cellular models at much lower concentrations than those inhibiting MAO or AChE. When ladostigil (1 mg/kg/day) was given for 6 months to 16-month-old rats it prevented the age-related increase in activated astrocytes and microglia in several hippocampal and white matter regions and increased proNGF immunoreactivity in the hippocampus towards the levels in young rats. Ladostigil also prevented the age-related reduction in cortical AChE activity and the increase in butyrylcholinesterase activity in the hippocampus, in association with the reduction in gliosis. The immunological and enzymatic changes in aged rats were associated with improved spatial memory. Ladostigil treatment had no effect on memory, glial or proNGF immunoreactivity in young rats. Early treatment with ladostigil could slow disease progression in conditions like Alzheimer's disease in which oxidative stress and inflammatory processes are present.     

老年学习记忆减退大鼠脑线粒体DNA缺失

目的和方法:测定老年大鼠脑mtDNA缺失型(以下简称缺失型),缺失mtDNA比例,探讨mtDNA缺失与老年性学习记忆减退的关系,为研究其分子机制提供基础资料。用Morris水迷宫将老年大鼠(24个月)筛选为老年学习记忆正常和学习记忆减退两组。用稀释PCR法测定大鼠大脑皮质、海马和小脑缺失型mtDNA比例。结果:青老年鼠大脑皮质、海马和小脑均存在mtDNA缺失,片段为4834bp;青年鼠的缺失比例约为0.00018%。老年记忆正常鼠上述3个脑区缺失型mtDNA缺失比例分别是青年鼠的6倍、6倍和2倍;老年记忆减退大鼠上述脑区的缺失型mtDNA比例分别是老年记忆正常鼠的2倍、1.8倍和3倍。结论:衰老时脑组织缺失型mtDNA增多,而老年学习记忆减退鼠较老年学习记忆正常鼠mtDNA缺失进一步成倍增加,表明相关脑区的mtDNA缺失在老年学习记忆减退的细胞分子机制中发挥重要作用。     

Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.     

Age-related changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat.

Enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were determined in the liver as well as several specific brain regions of young and old Fischer-344 rats of both sexes. In the liver of male rats, activities of CAT as well as Mn-SOD were lower, while activities of Cu Zn-SOD were higher in old (30-month-old) rats than in young (7-month-old) ones. Activities of total SOD as well as GSH Px were comparable for young and old male rat livers. In contrast to male rats, in female rat livers, activities of CAT were significantly higher in old (28-months-old) rats, while activities of Mn-SOD were slightly (but significantly) higher in old rat livers. In old male rats, activities of Mn-SOD were significantly higher than in young males in several specific regions of the brain (the substantia nigra (s. nigra), striatum, hippocampus) but lower in the cerebellum. In particular, SOD activities in s. nigra, striatum and hippocampus in old male rats were several fold higher than corresponding values in young male rats. Activities of Cu Zn-SOD were generally unchanged with age. Activities of CAT as well as GSH-Px (both Se-dependent and non-Se-dependent forms) were also relatively unaffected by age. In female rat brains, activities of Mn-SOD as well as those of others all remained mostly unaffected by aging, although there was a general tendency of slightly higher activities in most cerebral regions for Mn-SOD in old female rats. Thus, age-related changes of these antioxidant enzymes in the liver and brain are markedly sex dependent and some enzyme activities (such as CAT in the liver) change in an opposite direction with age. Changes of Mn-SOD in the brain were markedly region-specific in male rats. Results suggest that the significance of the changes of these antioxidant enzyme activities during aging needs to be carefully interpreted, taking into consideration the fact that changes are markedly variable depending on sex as well as the organs and brain regions examined.     

Translation fidelity in the aging mammal: studies with an accurate in vitro system on aged rats

The accuracy of poly(U) translation was measured in the post-mitochondrial supernatant from whole brain of 7- and 33-month-old Fischer 344 rats. Measurements were made: under in vitro conditions in which translation fidelity was similar to what is known about the accuracy of translation in vivo; and under stresses of varying Mg2+ concentrations (3-12 mM), pH (6.6-8.4), temperature (26-42 degrees C) and in the presence or absence of 2.4% ethanol. No significant difference could be detected between the responses of old and young extracts, the activities of their Phe- and Leu-tRNA synthetases, and their endogenous amounts of Phe-tRNA and Leu-tRNA, despite the fact that the rats studied corresponded in age (by actuarial criteria) to 90-year-old human beings. The accuracy of poly(U) translation was also studied: in liver and hippocampus extracts from 7- and 33-month-old rats; and in brain extracts from 3- and 29-month-old rats. The results were similar to those obtained in brain extracts from 7-month-old rats. Explanations are provided for the inconsistencies which exist in the literature regarding the effect of aging on the accuracy of protein synthesis. It is shown that the inconsistencies are likely to reflect inadequate methodology in three previous studies rather than biological diversity in the control of translation fidelity in aged animals.     

Age-related changes in glucocorticoid receptor binding and mRNA levels in the rat brain and pituitary.

Hypothalamic-pituitary-adrenal (HPA) activity under both basal and poststress conditions is often increased in the aged rat. This change has been associated with the loss of corticosteroid receptors in specific regions that mediate glucocorticoid negative feedback. In order to study the cellular basis for the loss of receptors, we measured glucocorticoid (type II corticosteroid) receptor binding and mRNA levels in pituitary and selected brain regions in rats at 6, 12, and 24 months of age. Receptor binding, measured using [3H]RU 28362, was stable in all regions (pituitary, hypothalamus, amygdala, and frontal cortex) except the hippocampus, where there was about a 40% decrease in binding capacity, with no change in the affinity of the receptor for RU 28362. The loss of receptors in the hippocampus was apparent in animals at 12 months of age, and binding was further decreased at 24 months. Glucocorticoid receptor mRNA levels were significantly higher in all regions at 12 months of age than at 6 months. By 24 months, however, receptor mRNA levels in most regions had returned to levels that were similar to those at 6 months of age. In contrast, glucocorticoid receptor mRNA levels in the hippocampus were significantly decreased at 24 months of age compared to levels at both 6 and 12 months of age. Thus, in general, variations in receptor mRNA levels parallel those in receptor binding in animals 6 and 24 months of age, with the hippocampus as the only region showing a significant loss of receptors and a decrease in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orbitofrontal dopaminergic dysfunction causes age-related impairment of reversal learning in rats

Reversal learning is a domain that involves cognitive flexibility and is defined as the ability to rapidly alter established patterns of behavior when confronted with changing circumstances. This function depends critically on the orbitofrontal cortex (OFC) in the prefrontal cortical (PFC) structure, which is among the most sensitive to the influences of aging, and impaired reversal learning is a common functional disturbance of aged brain. The present study was designed to clarify the precisely neurochemical basis of this impaired learning in rats. For this purpose, we first examined reversal learning in young (3-month-old) and aged (24-month-old) rats using a T-maze discrimination task. The ability of aged rats to learn initially a reward rule for a T-maze discrimination task was almost equal to that of young rats, suggesting that simple discrimination ability was normal in aged rats. However, the ability to learn a reversed rule in a subsequent task was markedly impaired in aged rats. In addition, aged rats had reduced dopaminergic transmission concomitant with attenuated tyrosine hydroxylase (TH) activity in the OFC. Moreover, age-related impairment of reversal learning was improved by an intra-OFC infusion of 30 ng, but not 10 ng, of the D1 receptor agonist SKF 81297. Increasing dose of SKF 81297 to 100 ng also improved the impairment, but this effect was weaker than that of 30 ng, indicating that the SKF 81297 response was an inverted “U” pattern. The maximum SKF 81297 response (30 ng) was abolished by the D1 receptor antagonist SCH 23390. Thus, age-related impairment of reversal learning was due to a D1 receptor-mediated hypodopaminergic mechanism in the OFC. This finding provides direct evidence showing the involvement of OFC dopaminergic dysfunction in the development of cognitive inflexibility during the normal aging process