Segment-specific changes with age in the expression of junctional proteins and the permeability of the blood-epididymis barrier in rats.

In aging Brown Norway rats, there is a striking increase in the number of halo cells in the epididymis; this reflects an activation of the immune system. As the blood-epididymis barrier should protect from immunological attack, we hypothesized that there would be changes in the structure and function of this barrier with age. To test this hypothesis, we assessed the immunocytochemical localization of occludin, ZO-1, and E-cadherin, as well as the lanthanum nitrate permeability of the blood-epididymis barrier, in the epididymides of Brown Norway rats aged 3, 18, and 24 mo. In the initial segment, occludin, ZO-1, and E-cadherin immunostaining was observed at the apico-lateral junction between principal cells in the 3-mo-old animals; with increasing age, occludin and ZO-1 reactivity decreased, while E-cadherin staining increased along the lateral membrane between principal cells. In the caput, corpus, and cauda epididymidis, occludin, ZO-1, and E-cadherin immunostaining showed segment-specific and age-dependent differences in their staining patterns. The most dramatic changes were seen in the corpus epididymidis with age; the intense E-cadherin cytoplasmic staining that was observed at 3 mo was absent by 24 mo, and no occludin or ZO-1 reactivity was observed in older animals. The greatest penetration of lanthanum nitrate across the blood-epididymis barrier and in the lumen was seen in the aging corpus epididymidis, while there was no barrier permeability in the initial segment or cauda epididymidis of the aged animals. Taken together, these data indicate that there are segment-specific decreases in the structural and functional integrity of the blood-epididymis barrier with age, most notably in the corpus epididymidis.     

The effect of age on lung structure in male BALB/cNNia inbred mice

Morphometric examination using light, scanning electron, and transmission electron microscopy was performed on the lungs from 32 inbred male BALB/ cNNia mice between 38 days and 28 months of age. Between 38 days and 9 months of age the changes were primarily those of growth. Alveolar multiplication and total elastic-fiber length appeared complete by 38 days of age. The major increase in the number of interalveolar pores occurred by 68 days, but there was a significant further increase from 68 days to 9 months of age. At 9 months, approximately 10% of the alveolar wall was formed by pores. Alveolar ducts, the cylindrical core of air central to alveolar mouths, increased more in diameter than length. Between 9 and 28 months the changes were attributed to aging and were different from those reported in humans and other species. Lung volume, alveolar surface area, and total volume of alveolar wall increased with age; there was no change in mean linear intercept and volume proportion of alveolar and alveolar duct air. Total area of pores increased with age, but their number and area fraction of the alveolar wall did not change. No transmission electron microscopic changes were seen in the alveolar walls. We speculate that the morphometric differences between our animals and those studied in other reports may reflect the fact that our animals were specific-pathogen-free animals and kept under protected circumstances.     

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450

The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.     

Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

Remarkable changes occur during aging in the testis and epididymis of the Brown Norway rat. A dramatic increase in the number of halo cells, which are present in the epididymal epithelium and originate from the immune system, is found in animals of increasing age. Halo cells have been postulated to be either lymphocytes or monocytes. We hypothesized that halo cells are a mixture of different immune cells and that their relative composition changes with age. To verify this hypothesis, markers for helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, and monocytes-macrophages were used to identify the major categories of immune cells in the epididymides of Brown Norway rats ranging in age from 3 to 24 mo. The numbers of immunocompetent cells in the epididymis were determined in relation to age, epididymal segment, and luminal content. We found that monocytes, helper T lymphocytes, and cytotoxic T lymphocytes belong to the population of halo cells. In addition, a segment-specific increase with age in the number of these immune cells was noted. Finally, we report a segment-specific recruitment of cytotoxic T lymphocytes and monocytes-macrophages in the epididymal epithelium of aged rats whose epididymal lumen contained few spermatozoa. We postulate that accumulation of damaged epithelial cells and antigens of germ cell origin, leaking through a dysfunctional blood-epididymis barrier, may contribute to the active recruitment of immune cells with age.     

Remodeling of neuronal membranes as an early response to deafferentation. A freeze-fracture study

The early effects of deafferentation on the postsynaptic membrane beneath the end bulb of Held in the anteroventral cochlear nucleus (AVCN) were studied with the freeze-fracture technique. Three distinct responses were seen on the external membrane leaflet after cochlear ablation. Within 12 h the number of nonaggregate particles increased 147% by the addition of new particles to the membrane. The increase in number of nonaggregate particles continued until 4 days after cochlear ablation. The other responses occurred later, after degenerative changes were present in the end bulb. Between 1 and 2 days after cochlear ablation, the number of perisynaptic aggregates surrounding the postsynaptic active zone decreased to 10% of normal numbers. By 4 days, all perisynaptic aggregates had disappeared from the membrane. Coated vesicles may be involved in removing these aggregates. Between 1 and 3 days, the number of junctional aggregates decreased, but the size of the aggregates increased, apparently as a result of coalescence of nearby junctional aggregates. The total number of particles in junctional aggregates in the membrane was not altered during the first 6 days after cochlear ablation. The three separate responses suggest the existence of at least three different types of intramembranous particles on the external leaflet of the principal cell membrane, with each type dependent upon different cues for its maintenance in the membrane.     

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.     

Aging neural progenitor cells have decreased mitochondrial content and lower oxidative metabolism

Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology.     

Aging of mouse eggs in vivo and in vitro

Morphological and cytochemical (acid phosphatase) changes associated with mouse ova and cumulus cells aged within the oviducts (in vivo) or in culture (in vitro; 1–24 hours postovulation) have been investigated. Structural alterations of cumulus cells were apparent immediately after ovulation and included nuclear pycnosis and cytoplasmic vacuolization. Nevertheless, approximately 30% of the cumulus masses examined contained cells that plated out when cultured and remained viable for up t o three days in vitro. From 12 t o 24 hours postovulation almost all cumulus cells of specimens aged in vivo showed signs of degeneration. Disruption of the meiotic spindle and an increase in acid phosphatase positive organelles were characteristic of in vivo and in vitro aging ova. The percentage of fragmented eggs obtained from super-ovulated (5 IU PMS followed by 5 IU HCG) mice approximately one and 24 hours postovulation was not significantly different. Eggs obtained from superovulated animals and aged in vitro for 24 hours yielded significantly more fragmented ova. Fragmented eggs were not obtained from cycling females on the morning of estrus. When such eggs were cultured in vitro for 24 hours the percent fragmentation was significantly lower than that for aged eggs obtained from super-ovulated mice. These results indicate that 1) similar morphological alterations occur among cumulus cells and eggs aged either in vitro or in vivo, 2) ova from superovulated mice do not constitute a homogeneous population and 3) the method of superovulation employed in this study induces the ovulation of a relatively large group of eggs that are susceptible to fragmentation when cultured in vitro.     

Aging and oxidatively damaged nuclear DNA in animal organs

Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies reporting a baseline level of damaged DNA that was fewer than 5 lesions/10⁶ dG showed that 21 of 29 studies reported age-associated accumulation of DNA damage. The standardized mean difference in oxidatively damaged DNA between the oldest and the youngest age groups was 1.49 (95% CI 1.03–1.95). There was no difference between age span, number of tested organs, statistical power, sex, strain, or breeding between the studies showing positive and null effects. Citation and publication bias seems to be a problem in the overall dataset, whereas it is less pronounced in the restricted dataset. There is compelling evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation.     

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.     

Aging alters the metabolic flux signature of the ER‐unfolded protein response in vivo in mice

Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPR^ER) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a flux “signature” of the UPR^ER in vivo in mouse liver was developed by inducing ER stress with tunicamycin and measuring rates of both proteome‐wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared with young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was seen only in the triglyceride fraction. These data indicate that aged mice have an exaggerated metabolic flux response to ER stress, which may indicate that aging renders the UPR^ER less effective in resolving proteotoxic stress.     

The spike reactions of the motor cortex neurons of old rabbits to specific stimuli

Spike reactions of motor cortex neurons to tactile and electrocutaneous stimulation of a forelimb were studied in aged (6-7-year old) rabbits. As compared with young adult animals, the neuronal reactions to afferent stimuli were rarely recorded in the motor cortex of aged rabbits (66.7 and 50%, respectively). The activation manifested in increasing firing rate over its spontaneous level was less intensive than in young animals. The neuronal reactions of aged animals were characterized by the slower activation with longer latencies and slower development of spike responses. The parameters of slow activation could be partly corrected by the iontophoretic application of acetylcholine to the soma region. Neuronal inhibition recorded in the motor cortex of aged rabbits was not markedly changed compared to inhibition reactions in young animals. It is suggested that impairment of the functional state of dendrites in aging is responsible for the changes observed.     

Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration

Increased excitability of principal excitatory neurons is one of the hallmarks of aging in the hippocampus, signifying a diminution in the number and/or function of inhibitory interneurons with aging. To elucidate this, we performed comprehensive GABA-ergic interneuron cell counts in all layers of the dentate gyrus and the CA1 and CA3 subfields, using serial sections from adult, middle-aged and aged Fischer 344 rats. Sections were immunostained for glutamate decarboxylase-67 (GAD-67, a synthesizing enzyme of GABA) and GAD-67 immunopositive interneurons were counted using an unbiased cell counting method, the optical fractionator. Substantial declines in the absolute number of GAD-67 immunopositive interneurons were found in all hippocampal layers/subfields of middle-aged and aged animals, in comparison with the adult animals. However, the counts were comparable between the middle-aged and aged groups for all regions. Interestingly, determination of the absolute number of interneurons using neuron-specific nuclear antigen (NeuN) expression in the strata oriens and radiatum of CA1 and CA3 subfields revealed an analogous number of interneurons across the three age groups. Furthermore, the ratio of GAD-67 immunopositive and NeuN positive interneurons decreased from adult age to middle age but remained relatively static between middle age and old age. Collectively, the results underscore that aging in the hippocampus is associated with wide-ranging decreases in the number of GAD-67 immunopositive interneurons and most of the age-related changes in GAD-67 immunopositive interneuron numbers transpire by middle age. Additionally, this study provides novel evidence that age-related reductions in hippocampal GAD-67 immunopositive interneuron numbers are due to loss of GAD-67 expression in interneurons rather than interneuron degeneration.     

Caspase remodeling of the spectrin membrane skeleton during lens development and aging

Terminal differentiation of lens fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging lens fiber cells are compressed into the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein alpha-spectrin to approximately 150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave alpha-spectrin to approximately 150-kDa fragments and beta-spectrin to approximately 120- and approximately 80-kDa fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.     

年龄与性别相关的大鼠视皮层树突形态变化

衰老会导致视觉功能的退化,但其潜在的神经机制尚不清楚。通过改良Golgi-Cox染色法,测定了青年雄性、青年雌性及老年雄性与老年雌性4组共20只大鼠视皮层的树突长度和树突棘密度,以研究年龄与性别对视皮层树突形态的影响。结果显示青年雄性大鼠视皮层顶树突、基树突、树突总长度均明显高于青年雌性大鼠,但这种性别差异在老年雌雄组之间并不显著,可能是由于在雄性组之间存在着明显的年龄相关性树突长度减少而在雌性组之间并不存在。青年雄性组的树突棘密度要明显高于青年雌性组,尽管衰老导致了青年雄、雌性组的树突棘密度均明显降低,但老年雄、雌性组的树突棘密度并无显著差异,这可能是由于雄性组的年龄相关性树突密度降低程度要远大于雌性组。由此可见衰老确实能导致视皮层树突形态的退化,这可能是老年性视觉功能衰退的潜在神经机制,但这种退化可能具有一定的性别差异。     

Degeneration of Axotomized Projection Neurons in the Rat dLGN: Temporal Progression of Events and Their Mitigation by a Single Administration of FGF2

Removal of visual cortex in the rat axotomizes projection neurons in the dorsal lateral geniculate nucleus (dLGN), leading to cytological and structural changes and apoptosis. Biotinylated dextran amine was injected into the visual cortex to label dLGN projection neurons retrogradely prior to removing the cortex in order to quantify the changes in the dendritic morphology of these neurons that precede cell death. At 12 hours after axotomy we observed a loss of appendages and the formation of varicosities in the dendrites of projection neurons. During the next 7 days, the total number of dendrites and the cross-sectional areas of the dendritic arbors of projection neurons declined to about 40% and 20% of normal, respectively. The response of dLGN projection neurons to axotomy was asynchronous, but the sequence of structural changes in individual neurons was similar; namely, disruption of dendrites began within hours followed by cell soma atrophy and nuclear condensation that commenced after the loss of secondary dendrites had occurred. However, a single administration of fibroblast growth factor-2 (FGF2), which mitigates injury-induced neuronal cell death in the dLGN when given at the time of axotomy, markedly reduced the dendritic degeneration of projection neurons. At 3 and 7 days after axotomy the number of surviving dendrites of dLGN projection neurons in FGF-2 treated rats was approximately 50% greater than in untreated rats, and the cross-sectional areas of dendritic arbors were approximately 60% and 50% larger. Caspase-3 activity in axotomized dLGN projection neurons was determined by immunostaining for fractin (fractin-IR), an actin cleavage product produced exclusively by activated caspase-3. Fractin-IR was seen in some dLGN projection neurons at 36 hours survival, and it increased slightly by 3 days. A marked increase in reactivity was seen by 7 days, with the entire dLGN filled with dense fractin-IR in neuronal cell somas and dendrites.