Cells In Young Animals Research Articles

Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction.

Senescent cells, which accumulate in organisms over time, contribute to age-related tissue decline. Genetic ablation of senescent cells can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness. While small-molecule drugs that eliminate senescent cells ('senolytics') partially replicate these phenotypes, they require continuous administration. We have developed a senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting the senescence-associated protein urokinase plasminogen activator receptor (uPAR), and we previously showed these can safely eliminate senescent cells in young animals. We now show that uPAR-positive senescent cells accumulate during aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti-uPAR CAR T cells improves exercise capacity in physiological aging, and it ameliorates metabolic dysfunction (for example, improving glucose tolerance) in aged mice and in mice on a high-fat diet. Importantly, a single administration of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.

Tracking of Prosaposin, a Saposin Precursor, in Rat Testis.

We tracked prosaposin (PSAP), a trophic factor, using an antibody specific to its proteolytic portion and an antibody to sortilin that traffics PSAP only to the lysosome. Immunostaining revealed that PSAP was distributed mainly on the basal side of seminiferous tubules, where many Sertoli cells and pachytene spermatocytes contained PSAP and its distribution differed depending on the stage of the spermatogenic cycle. The PSAP-sortilin complex was sorted to large lysosomes in the basal cytoplasm of Sertoli cells, where it may be processed into saposins. In contrast, in the thinner apical cytoplasm of Sertoli cells, PSAP in small lysosomes was transported to the apical side around sperm heads or into the lumen for secretion. The results of in situ hybridization analyses suggested that immature tubular cells in young animals produce PSAP to self-stimulate proliferation. However, in adults, not only Sertoli cells but also pachytene spermatocytes produce and secrete PSAP around germ cells or into the tubular lumen to stimulate cell proliferation or differentiation in a paracrine or autocrine manner. In summary, PSAP is not only a precursor of lysosomal enzymes but also a pivotal trophic factor in organogenesis in the immature testis and spermatogenesis in the mature testis.

Short-term fenofibrate treatment improves ultrastructure of hepatocytes of old rats.

Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals. Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1. The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals. The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.

Depletion of Mature B Cells By Anti CD20-Directed Antibody Induces Trabecular Bone Loss in Young Mice: An Alert for Clinicians?

Targeted B cell therapy by anti-CD20 antibodies has revolutionized the treatment of B cell malignancies, immune cytopenias as well as connective tissue diseases. A growing body of evidence links B cell homeostasis and bone metabolism, including reports demonstrating severe bone loss in transgenic mice lacking IgM+ B cells. Here, we addressed the hypothesis that pharmacological depletion of mature B cells induces bone loss in an age-dependent manner.Ten-week and one-year old C57BL/6J female mice were treated with 150µg and 250µg, respectively, of mouse anti-mouse CD20 (hereafter αCD20), administered in two equal doses, two weeks apart. Four weeks after the first injection, distal femurs and L3 vertebrae (old mice only) were subjected to bone morphometric analysis using µCT. The efficiency of B cell depletion as well as immunophenotyping of the bone marrow (BM) and spleen were assessed by flow cytometry. We used ELISA to measure serum markers of bone formation (N-terminal propeptide of procollagen 1, P1NP)and bone resorption (tartrate-resistant acid phosphatase 5b, TRAcP 5b). The expression of receptor activator of nuclear factor κ-B ligand (RANKL), osteoprotegerin (OPG) and interleukin 7 (IL-7) in both BM containing bone fragment (BM-BF) and spleen was analyzed by means of RT-qPCR. To investigate the osteoclastogenic potential of various BM B cell subsets, total BM cells from wild-type untreated mice were flow-sorted and subjected to an osteoclastogenesis assay in RANKL- and macrophage colony stimulating factor (M-CSF)-containing medium.In young mice with >90% splenic B cell depletion (5 out of 7), αCD20 treatment (compared to diluent controls) induced a significant trabecular bone loss, reflected by 25% decrease in bone mineral density (30.1±2.5 vs 40±3.7 mgHA/cm3), 44% decrease in connectivity density (10.3±1.5 vs 18.9±2.8 mm-3), 13% reduction in trabecular number (2.8±0.13 vs 3.24±0.13 mm-1), and corresponding 14% increase in trabecular separation (p<0.05 for all parameters, n=5-7). However, no effect of αCD20 on bone mass was noted in the old mice (neither in femurs nor in L3 vertebrae, n=6 in each group).αCD20 treatment induced a compensatory increase in BM pro-B cells in young animals (2.8±0.5% vs 1.8±0.1%, p<0.05) which was even more pronounced in old ones. However, only the former exhibited a 2.5-fold increase in M-CSF receptor (CD115) positivity of this population (p<0.05). In that respect, our osteoclastogenic assays showed that among various BM B cell populations, only CD115+ pro-B cells could give rise to functional osteoclasts (18±6.5% OC area vs none). Moreover, serum P1NP levels were significantly lower in the young but not in the old αCD20-treated animals as compared to their corresponding diluent controls (48.2±8.7 ng/mL vs 74.5±4.6 and 27.9±8.2 ng/mL vs 30.3±10.8, respectively), indicating decreased bone formation in the former. αCD20 treatment did not affect the levels of TRAcP 5b, a surrogate marker of the total osteoclast number. Following B cell depletion, the expression of IL-7, known to induce bone loss, was upregulated in both BM-BF and spleen by the magnitude of 37% and 50%, respectively (p<0.05). Yet, the expression of RANKL and OPG in BM-BF and in spleen, were similar in control and in the αCD20 treatment groups.The current study demonstrates for the first time the adverse skeletal outcomes of pharmacological depletion of mature B cells, conceivably fueled by B cell-derived osteoclastogenesis in the presence of decreased bone formation. Since many patients treated with clinically available anti-CD20 antibodies already have other risk factors for bone loss, e.g. female gender and glucocorticoid therapy, these data also have immediate clinical implications. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells.

Non-mammalian vertebrates can restore their auditory and vestibular hair cells naturally by triggering the regeneration of adjacent supporting cells. The transcription factor ATOH1 is a key regulator of hair cell development and regeneration in the inner ear. Following the death of hair cells, supporting cells upregulate ATOH1 and give rise to new hair cells. However, in the mature mammalian cochlea, such natural regeneration of hair cells is largely absent. Transcription factor reprogramming has been used in many tissues to convert one cell type into another, with the long-term hope of achieving tissue regeneration. Reprogramming transcription factors work by altering the transcriptomic and epigenetic landscapes in a target cell, resulting in a fate change to the desired cell type. Several studies have shown that ATOH1 is capable of reprogramming cochlear non-sensory tissue into cells resembling hair cells in young animals. However, the reprogramming ability of ATOH1 is lost with age, implying that the potency of individual hair cell-specific transcription factors may be reduced or lost over time by mechanisms that are still not clear. To circumvent this, combinations of key hair cell transcription factors have been used to promote hair cell regeneration in older animals. In this review, we summarize recent findings that have identified and studied these reprogramming factor combinations for hair cell regeneration. Finally, we discuss the important questions that emerge from these findings, particularly the feasibility of therapeutic strategies using reprogramming factors to restore human hearing in the future.

ЧУТЛИВІСТЬ ЕРИТРОЦИТІВ ЩУРІВ РІЗНОГО ВІКУ ДО СТРЕСОВОГО ВПЛИВУ

In this work age features of 1- and 12-month rats erythrocytes sensitivity to hypertonic conditions of environment after their modification by hemin. It has been shown that the level of hemolytic damage of erythrocytes in both 1-month and 12-month rats depends on the time of incubation in hypertonic solutions of sucrose and their concentration. For incubations for 2, 10, 30, and 60 minutes significant increase in damage occurs at concentrations of 0.5–0.8 M (8–10 times). The analysis of cell damage, depending on the concentration of sucrose at the same incubation time, showed that for 10, 30, and 60 minutes it is possible to observe the initial increase in sensitivity, achievement of maximum (10 min – 0.8 M, 30 min – 0.7 M, 60 min – 0.6 M) and further reduction erythrocytes damage in both 1-month and 12-month rats. It was found that gemin treatment increases the level of hypertonic damage of erythrocytes after incubation in sucrose environments for 1 month old rats. At the same time, the maximum difference in the sensitivity of treated and control cells is observed at incubation time of 10 min and 30 min (33±4 % and 18±2 %, 56±4 % and 44±3 % respectively). The level of erythrocyte damage in 12-month-old rats in these conditions does not change. For 10 and 30 minutes incubation (21±3 % and 15±2 %, 27±3 % and 23±2 % respectively). Since hemin modifies protein 4.1, the data indicate the importance of 4.1-spectrum protein bonds to support the mechanical stability of red blood cells in young animals. Overall level of hemolytic damage of erythrocytes for 12-month-old animals is lower for both control and modified cells for all concentrations of sucrose. The percentage of hemolysis is reduced from two to three times. Thus, erythrocytes of 1-month and 12-month-old rats differ significantly in sensitivity, both to modification by hemin, and to the stressful effects of hypertonic sucrose solutions. Senior animal cells exhibit a lower level of damage in both cases. This can be explained by the lower availability of macromolecular complexes that support the stability of the membrane, for modifying the influence of hemin and low ionic strength media, as well as the different distribution of strain on macromolecular structures that maintain the integrity and mechanical stability of the membrane in animals of all ages.

Bone Marrow Cells Obtained from Old Animals Differ from the Young Animals Cells in Their Ability to Divide and in Response to the Presence of Liver Fibrosis in Primary Culture

Dynamics of bone marrow cells number (BMC) in the primary culture isolated from young (3 months) and old (20 months) Wistar rats was investigated. Proliferative activity of BMC of old animals was 2 times higher than that of young animals in the primary culture. Such superiority of the proliferative activity of BMC in the primary culture obtained from old animals is associated with the ability to actively divide lymphocytes and longer “lifespan” of segmented neutrophils obtained from old animals. It should be noted, that the lymphocytes of young animals did not proliferate in the primary culture. The content of intracellular calcium in BMC in the cells of old animals was 3 times higher compared with cells of young animals, which revealed the relationship of intracellular calcium and proliferative activity of BMC. Induction of liver fibrosis led to an increase in the lymphocyte content in young animals by 167%, and in old ones only by 26%, while the lymphocytes of young animals acquired the ability to proliferate in the primary culture. It has been suggested that such differences in the behavior of BMC in primary culture obtained in young and old animals reflect differences in the BMC microenvironment of young and old animals, which leads to changes in the epigenetic-metabolic characteristics of BMC.

Donor Age Affects Behavior and Sensibility of Bone Marrow Cells to Copper Ions in Primary Culture

In the primary culture of bone marrow cells obtained from young (3-month-old) and old (20-month-old) rats, the change in the number of cells from day 0 to day 4, the pattern of cell morphotypes, and the lifespan of myelocytes, metamyelocytes, and stab and segment neutrophils was studied. It was shown that the number of bone marrow cells obtained from old animals (20-month-old) in the primary culture increased faster than the number of bone marrow cells in young animals (3-month-old). The presence of Copper-induced liver fibrosis in animals had a different effect on the rate of increase in the number of bone marrow cells obtained in young and old animals. The administration of 4 and 8 mmol of CuSO4 ⋅ 5H2O into the culture of bone marrow cells in young and old animals caused a dose-dependent inhibition of proliferative processes in the cells of young animals. When copper ions were administered into the culture of bone marrow cells obtained from old animals, the inhibition of proliferation was less pronounced than for young animals, and a concentration of 8 mmol of CuSO4 ⋅ 5H2O inhibited proliferation to a lesser extent than 4 mmol of CuSO4 ⋅ 5H2O. The presence of liver fibrosis in animals accelerated the process of bone marrow cell death in the primary culture in young and old animals. However, this effect was more pronounced in young animals. It is suggested that bone marrow cells undergo such epigenetic changes, which change their functional properties, during ontogeny.

Effect of gold and silver nanoparticles on the morpho-functional state of the epididymis and prostate gland in rats

Metals are widely used in modern medicine: iron, copper, zinc, vanadium, titanium – all of them are vital for treatment of different diseases. Recently a new field of medical technology has emerged, which focuses on the biomedical application of metallic nanoparticles, with a particular interest in a gold and silver-based materials. These structures are already used for photothermal anticancer therapy, drug delivery, bioimaging, radiosensitizers and as drugs themselves. Despite the wide usage of nanoparticles, we still don’t know much about the toxicity of nanomaterials. Nanotoxicological studies are mainly carried out in vitro, but in vivo effects are still elusive. Hence, we focused on the reproductive toxicity of gold and silver nanosized particles. Spherical 10–15 nm gold and silver nanoparticles were synthesized through the reduction of sodium tetrachloroaurate (III) and silver nitrate respectively with ascorbic acid in the presence of sodium polyphosphate as a coating and stabilizing agent. Next, these particles were administered intraperitoneally to the young and adult animals (1- and 6-months old respectively) at 1 mg/kg dose for 10 days. As quantitative markers of functional activity, we used the diameter of epididymal tubules, height and the nuclear cross-section of epididymal epitheliocytes and relative volume of the prostatic epithelium. We showed that intraperitoneal administrations of nanogold to young animals caused no significant histological changes, although we found a decrease in the nuclear cross-sectional area of epididymal epitheliocytes. At the same time, nanogold caused more morphometric changes in adult animals. Similar results were obtained from the nanosilver groups. Silver nanoparticles caused an observable decrease of sperm quantity in the lumen of epididymal tubules with a simultaneous increase in the number of extraepididymal cells in young animals. Morphometric parameters of the epididymis and prostate also decreased. Administration of nanosilver to adult animals also downregulated the morpho-functional state of both organs, although no histological changes were found. We showed that both nanogold and nanosilver can cause adverse effects on the functional activity of the epididymis and prostate of rats. It is worth mentioning that silver nanoparticles were generally more toxic than the gold ones, which correlates with their known mechanism of cellular action. Although the exact mechanisms of gold and silver action require further study, our results are useful for practical usage of nanomaterials in biomedical and clinical practice.

Atoh1 Directs the Formation of Sensory Mosaics and Induces Cell Proliferation in the Postnatal Mammalian CochleaIn Vivo

Hearing impairment due to the loss of sensory hair cells is permanent in humans. Considerable interest targets the hair cell differentiation factor Atoh1 as a potential tool with which to promote hair cell regeneration. We generated a novel mouse model to direct the expression of Atoh1 in a spatially and temporally specific manner in the postnatal mammalian cochlea to determine the competency of various types of cochlear epithelial cells for hair cell differentiation. Atoh1 can generate cells in young animals with morphological, molecular, and physiological properties reminiscent of hair cells. This competency is cell type specific and progressively restricted with age. Significantly, Atoh1 induces ectopic sensory patches through Notch signaling to form a cellular mosaic similar to the endogenous sensory epithelia and expansion of the sensory mosaic through the conversion of supporting cells and nonautonomous supporting cell production. Furthermore, Atoh1 also activates proliferation within the normally postmitotic cochlear epithelium. These results provide insight into the potential and limitations of Atoh1-mediated hair cell regeneration.

Photometric and fluorimetric studies of protomitochondria from liver of young and adult rats

Protomitochondria (PRM) — young organelles — were isolated from liver cells of young (1 month) and adult (9 months) rats. PRM were characterized by photometric and fluorimetric methods. It was found that the number of PRM of various diameters in young and adult rats differs. PRM and mitochondria (MCH) from young and adult rats differ significantly. It was detected by fluorescence of 1-anilinonaphtalene-8-sulfonate (ANS), indicating a different number of membrane binding sites for this probe. An increase of the cytochrome content was observed during ripening of PRM to MCH, more pronounced in young animals. In contrast, the amount of cytochromes in MCH decreases during maturation of young rats to adult rats. The obtained data suggest that massive transformation of PRM to MCH in the cells of young animals takes place, similar to the transformation in the cells of adult animals observed earlier.

CD4+ lymphocytes modulate prostate cancer progression in mice

Chronic inflammation contributes to the development of prostate cancer in humans. Here, we show that male Apc(Min/+) mice also develop prostate carcinoma with increasing age, mimicking that seen in humans in their 5th or 6th decade of life. Proinflammatory cytokines were significantly linked with cancer and increasing age in our mouse model; however, prostate and bowel tissues lacked evidence of inflammatory cell infiltrates other than mast cells. Lymphocytes protected against cancer, and protection from prostate cancer resided in antiinflammatory CD4(+)CD25(+) regulatory (T(REG)) cells that downregulated inflammatory cytokines. Supplementation with syngeneic T(REG) cells collected from wild-type mice reduced the levels of interleukin (IL)-6 (p < 0.05) and IL-9 (p < 0.001) and lowered prostate cancer risk (p < 0.05). Depletion of CD25(+) cells in 2-month-old animals increased the expression of IL-6 (p < 0.005) within prostate and increased the frequency of high-grade prostatic intraepithelial neoplasia (p < 0.05) and microinvasive prostatic carcinoma (p < 0.05) in dorsolateral prostate. Depletion of CD25(+) cells in young animals also increased the frequency of intestinal cancer in Min mice. Taken together, chronically elevated proinflammatory cytokines promoted carcinoma in Apc(Min/+) mice. T(REG) lymphocytes downregulated inflammation-associated carcinogenic processes and contributed to immune and epithelial homeostasis.

Immunohistochemical Studies of Stellate Cells in Experimental Cholestasis in Newborn and Adult Rats

BACKGROUND AND AIMSAlthough there is much known about liver diseases, some aspects remain unclear, such as the nature of the differences between the diseases observed in newborn infants and those in adults. For example, how do newborns respond to duct epithelial cell injury? Do the stellate cells in newborns respond similarly to those in adults during biliary obstruction?METHODSNinety newborn Wistar rats aged six days, weighing 8.0 – 13.9 g each, and 90 adult rats weighing 199.7 – 357.0 g each, were submitted to bile duct ligation. After surgery, they were randomly divided and sacrificed on the 3rd, 5th, 7th, 14th, 21st or 28th day post-bile duct ligation. Hepatic biopsies were obtained and immunohistochemical semi-quantification of desmin and α-SMA expression was performed in hepatic stellate cells and in myofibroblasts in the portal space, and between the portal space and the liver lobule.RESULTSDesmin expression in the myofibroblast cells post-bile duct ligation was higher in young rats, reaching its peak level in a shorter time when compared to the adult animals. The differences between the groups for α-SMA expression were less significant than for desmin.CONCLUSIONSThese findings indicate that there is an increase in the number of collagen-producing myofibroblast cells in young animals, suggesting that there is more intense fibrosis in this population. This finding may explain why young animals with bile duct obstruction experience more intense portal fibrosis that is similar to the pathology observed in the livers of newborns with biliary atresia.

A diacylglycerol lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an age-dependent manner

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

Evidence that age‐related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis approximately 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging.

Estrogen and aging affect the subcellular distribution of estrogen receptor-alpha in the hippocampus of female rats.

Estrogen replacement increases both the number of dendritic spines and the density of axospinous synapses in the hippocampal CA1 region in young rats, yet this is attenuated in aged rats. The estrogen receptor-alpha (ER-alpha) is localized within select spines of CA1 pyramidal cells in young animals and thus may be involved locally in this process. The present study investigated the effects of estrogen on the ultrastructural distribution of ER-alpha in the CA1 of young (3-4 months) and aged (22-23 months) Sprague Dawley rats using postembedding immunogold electron microscopy. Within dendritic spines, most ER-alpha immunoreactivity (IR) was seen in plasmalemmal and cytoplasmic regions of spine heads, with a smaller proportion within 60 nm of the postsynaptic density. In presynaptic terminals, ER-alpha-IR was clustered and often associated with synaptic vesicles. Significant effects of both aging and estrogen were observed. Quantitative analysis revealed that nonsynaptic pools of ER-alpha-IR within the presynaptic and postsynaptic compartments were decreased (35 and 27%, respectively) in the young estrogen-replaced animals compared with those that received vehicle. Such localized regulation of ER-alpha in response to circulating estrogen levels might directly affect synaptic signaling in CA1 pyramidal cells. No estrogen treatment-related differences were observed in the aged animals. However, 50% fewer spines contained ER-alpha in the aged compared with young hippocampus. These data suggest that the decreased responsiveness of hippocampal synapses to estrogen in aged animals may result from age-related decrements in ER-alpha levels and its subcellular localization vis-à-vis the synapse. Such a role for spinous ER-alpha has important implications for age-related attenuation of estrogen-induced hippocampal plasticity.

Estrogen and Aging Affect the Subcellular Distribution of Estrogen Receptor-α in the Hippocampus of Female Rats

Estrogen replacement increases both the number of dendritic spines and the density of axospinous synapses in the hippocampal CA1 region in young rats, yet this is attenuated in aged rats. The estrogen receptor-alpha (ER-alpha) is localized within select spines of CA1 pyramidal cells in young animals and thus may be involved locally in this process. The present study investigated the effects of estrogen on the ultrastructural distribution of ER-alpha in the CA1 of young (3-4 months) and aged (22-23 months) Sprague Dawley rats using postembedding immunogold electron microscopy. Within dendritic spines, most ER-alpha immunoreactivity (IR) was seen in plasmalemmal and cytoplasmic regions of spine heads, with a smaller proportion within 60 nm of the postsynaptic density. In presynaptic terminals, ER-alpha-IR was clustered and often associated with synaptic vesicles. Significant effects of both aging and estrogen were observed. Quantitative analysis revealed that nonsynaptic pools of ER-alpha-IR within the presynaptic and postsynaptic compartments were decreased (35 and 27%, respectively) in the young estrogen-replaced animals compared with those that received vehicle. Such localized regulation of ER-alpha in response to circulating estrogen levels might directly affect synaptic signaling in CA1 pyramidal cells. No estrogen treatment-related differences were observed in the aged animals. However, 50% fewer spines contained ER-alpha in the aged compared with young hippocampus. These data suggest that the decreased responsiveness of hippocampal synapses to estrogen in aged animals may result from age-related decrements in ER-alpha levels and its subcellular localization vis-à-vis the synapse. Such a role for spinous ER-alpha has important implications for age-related attenuation of estrogen-induced hippocampal plasticity.

Aging confers different sensitivity to the neurotoxic properties of unconjugated bilirubin.

The pathogenesis of bilirubin encephalopathy appears to result from accumulation of unconjugated bilirubin (UCB), which, in turn, may cause mitochondrial perturbation, release of intermembrane proteins, and, ultimately, cell death. Aging imparts to cells a different susceptibility to this toxic stimulus, as neonates are particularly vulnerable to the accumulation of UCB in the CNS. In this paper, we further characterize UCB-induced toxicity in isolated neuronal and glial cells according to age in culture. In addition, we investigate sensitivity of mitochondria derived from young and old rats to UCB-induced membrane permeabilization and, finally, evaluate whether age-dependent changes in UCB toxicity are accompanied by alterations in the mitochondrial content of cytochrome c. The results showed that UCB is more toxic to immature neural cells after 4-5 d in culture (p < 0.001), whereas neurons were more sensitive than astrocytes (p < 0.05). In fact, approximately 40% of cells were apoptotic in immature cultures compared with 20% in mature cultures. Unexpectedly, mitochondrial swelling and subsequent efflux of cytochrome c induced by UCB were 2-fold greater in organelles derived from older rats (p < 0.01). In conclusion, UCB toxicity of isolated rat neuronal and glial cells is modulated by age in culture in that immature cells are more susceptible. Mitochondria derived from younger rats are nevertheless more resistant to membrane permeabilization and cytochrome c release induced by UCB. The data indicate that the cells of young animals are relatively resistant to UCB toxicity, through a protective mechanism at the mitochondrial level; however, this is not sufficient to prevent apoptosis of cells in the young animal. Thus, although playing a role, direct mitochondrial injury may not be the sole mechanism of UCB cytotoxicity.

Role of Stromal Cells in Protecting Young and Aged B-Lineage Precursors from Dexamethasone-Induced Apoptosis

INTRODUCTION. B-lymphopoeisis is a balance between expansion and cell death. Bone marrow stromal cells support B-lymphopoeisis through direct cell-cell contact and by the production of growth factors, most notably IL-7, IGF-1, and SCF (1). Exposure to dexamethasone either in vivo or in vitro will cause the death of B-cells in early stages of development by inducing apoptosis. B-cell precursors appear to be protected from dexamethasone-induced apoptosis when co-cultured with the stromal cell line BMS2 (2). IL-7 is a possible anti-apoptotic factor since an increase in percentages of apoptotic B-cell precursors is seen in mice deficient in IL-7 production (3). However, even in IL-7 knockout mice, significant numbers of precursors still survive. Based on these findings, we hypothesized that IL-7 is the stromal derived mediator that protects B-cell precursors from dexamethasoneinduced apoptosis and the protective effects of IL-7 may be augmented by the growth factors IGF-1 and SCF. Furthermore, while IL-7 has been shown to induce proliferation in primary cultures of B-lymphocytes from young animals, this effect is not seen in primary cultures of Blymphocytes from aged animals. It is possible that, in aged animals, IL-7 acts to protect Blymphocytes from apoptosis in a manner similar to that observed with cells in young animals.

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats.

Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl- conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2-3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl- current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2-3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl- current and the ionomycin-activated Cl- current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl- channels, rather than to a deficiency of a cellular mediator.