NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.     

ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

Age Peculiarities of the Calpain/Calpastatin Cerebral System in Rats: Relation to the Hypothesis of Brain Aging

We studied age-related changes in the activity of calpain, those in the activity of its endogenous inhibitor calpastatin, and the ratio of these indices in the brain of rats of four age groups (2-3 weeks, 2-3, 5-6, and 24 months). The activity of calpain was estimated using FITC casein as the substrate. In a soluble fraction of the brain homogenate, the enzyme activity in general increased with age. In mature rats (5 to 6 months old), this index exceeded 3.65 times the corresponding index in juvenile (2 to 3 weeks old) animals, while in old animals this index somewhat decreased. In the fraction obtained after separation of calpain from other components using DEAE-cellulose chromatography, the age-related trend toward an increase in the activity of calpain was preserved, but it was much more moderate. The activity of calpastatin demonstrated an opposite direction of age changes: it was the maximum in 2-3-week-old animals and gradually decreased (by 27%) in old rats. We also found that the efficacy of inhibitory action of calpastatin in the cerebral tissue with respect to the activity of calpain was, as a rule, redundant. In this case, the ratio of inhibitor/enzyme activities decreased with age; this index was 1.65, 1.33, 1.1, and 1.0 or less in 2-3-week-old, 2-month-old, mature, and old animals. Therefore, we found that the intensity of calpain-mediated proteolysis in the rat brain increases from the juvenile period to the mature age and somewhat decreases in old individuals. Such alterations are developed at the expense of both an increase in the activity of the enzyme and weakening of the action of its inhibitor (calpastatin).     

Effect of Age on Vitamin E Concentrations in Various Regions of the Brain and a Few Selected Peripheral Tissues of the Rat, and on the Uptake of Radioactive Vitamin E by Various Regions of Rat Brain

Abstract: The concentrations of tocopherols in selected areas of the brains and a few peripheral tissues of 3-, 14-, and 30-month-old male Fischer 344 rats were determined by a high-performance liquid chromatographic method. Throughout the time period studied, α-tocopherol was the only tocopherol detected in the brain. Concentrations of α-tocopherol increased significantly with age in medulla and spinal cord whereas no such change was seen in other brain areas. Among the peripheral tisues, total tocopherol concentrations increased with age in the liver and adipose tissue while no significant changes were observed in the heart. The pattern of uptake of radioactive α-tocopherol from the serum by the various areas of the brain was similar for the 3-and 14-month-old animals even though the brains from the 14-month-old animals took up less of the radioactive compound. Measurable amounts of tocopherol esters were not present in the tissues of the 30-month-old animals.     

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.     

Plasma and pituitary concentrations of LH, FSH and prolactin in aged female C57BL/6 mice

Plasma and pituitary concentrations of LH, FSH and prolactin were determined by radioimmunoassay in 2-month-old (young) and 16-20-month-old (old) C56BL/6 mice. There were no statistical differences in hormonal levels between aged females in oestrus (those exhibiting a copulatory plug) and those in constant dioestrus. In the old females plasma levels of LH (P < 0.002) and FSH (P < 0.001) were significantly elevated, while levels of prolactin (P < 0.001) were significantly depressed when compared with those from young animals. Pituitary homogenates from old females also contained more gonadotrophins (P < 0.001) and less prolactin (P < 0.001) than those of the young females. A radioreceptor assay utilizing a plasma membrane of luteinized rat or mouse ovaries indicated that LH from 2-month-old animals bound better to ovarian receptors (P < 0.05) than did LH from old mice, although radioimmunoassay of the same samples gave higher (P < 0.01) plasma LH levels for the old mice. Since the radioreceptor assay is considered to be a more sensitive test for biologically active LH, the results from these two types of assays suggest that there may be an alteration in the mouse LH molecule with age.     

Phospholipid metabolism in neuronal and glial cells during aging

The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.     

Structural changes in the hepatocytes of old rats with posthemorrhagic anemia

Using light and electron microscopy, and morphometry, it has been demonstrated that 15 min and 2.5 h after a single blood loss (2% of the body mass) organelle disintegration and destruction are more marked and preserved in hepatocytes of old (25-month-old) rats, as compared to adult (8-month-old) animals. Regenerative processes in adult rats lead to hepatocyte structure normalization already by the 10th hour after the experiment was started, while in old rats disseminated fatty dystrophy of hepatic cells is observed even on day 7. Thus, hepatocytes of old animals are more susceptible to blood-loss-induced disturbances, with the regenerative processes in them being less marked and slower than in adult animals.     

The influence of alpha-fluoromethylhistidine on the regional brain histamine and plasma corticosterone levels in aging

alpha-Fluoromethylhistidine, a histidine decarboxylase inhibitor, induced a significant depletion in the hypothalamic, midbrain, and cortical brain histamine amounts in 12- and 3-month-old rats. In all three brain regions the most evident depletion occurred 2 h after treatment. In both groups of rats midbrain histamine levels returned to control values 6 h after treatment; however, hypothalamic histamine depletion was still significant and more evident in the old than in the young animals. Cortical brain histamine also remained significantly depleted in old rats, but returned to control values in young animals 6 h after alpha-fluoromethylhistidine treatment. These results suggest that old rats show a slower rate of new histamine synthesis in the cortex and hypothalamus. Regional brain histamine depletion was associated with a very significant decrease in plasma corticosterone levels, which indicates that brain histamine-corticosterone interactions do occur.     

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.     

Effects of dexamethasone on insulin receptor in aging

The aim of this study was to examine the effects of dexamethasone (Dex) on functional properties of the rat insulin receptor (IR). Male Mill Hill hooded rats, 3, 6, 12, 18 and 21 months old, were injected with Dex (4 mg/kg) and rat liver and erythrocytes were used for experiments 18 h after Dex administration. Treatment with Dex lowered the specific binding (SB) of insulin (INS) in the liver of 3- and 18-month-old rats and concentration of INS binding sites (N1, N2) and the dissociation constant of low-affinity binding sites (Kd2) in the liver of 6- and 18-month-old rats. In addition, Dex treatment lowered the liver IR protein level in all analyzed groups, except 21-month-old rats where it remained unchanged, but raised the IR mRNA level in 18-month-old rats. In erythrocytes, treatment with Dex decreased SB and Kd2 (in animals 3 and 6 months old) and N1 (in ones 3 and 18 months old). Following Dex treatment, the INS plasma level increased (in rats 3, 18 and 21 months old), while glucose (Glu) concentration increased in 3 and 12 months old, but decreased in 6- and 21-month-old rats. In summary, Dex exerts the strongest effect on the erythrocyte IR of 3- and 6-month-old rats and the hepatic IR of 18-month-old rats. IR in both tissues is almost insensitive to Dex in 12- and 21-month-old rats. The pattern of age-related changes of IR induced by Dex does not correlate with changes of plasma Glu and INS.     

Age characteristics of acid-base equilibrium and the blood coagulation system in starvation]

The state of the acid-base balance and of the blood coagulation system was studied in starvation of young (5-6-month-old) and old (24-26-month-old) male rats during starvation. The periods of the maximal changes coincided in both systems; in the young animals the onset of the acidotic crisis and hypercoagulation development occurred earliear and were more pronounced. Old animals proved to be more resistant to the conditions of starvation and died later than the young ones.     

Labeling of RNA in young and adult rat brain: Evidence for different RNA processing

The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.     

Effect of Maharishi Amrit Kalash an ayurvedic herbal mixture on lipid peroxidation and neuronal lipofuscin accumulation in ageing guinea pig brain

The effects of ayurvedic herbal mixture Maharishi Amrit Kalash(MAK) were studied on brain lipid peroxidation, oxygen consumption, and lipofuscin accumulation in 10 months and 32 months old guinea pigs. Brain regions studied were cerebral cortex, hypothalamus, cerebellum and spinal cord. Parameters assessed were lipid peroxidation, oxygen consumption, and lipofuscin accumulation. The endogenous lipid peroxide was found to be increased significantly (P < 0.05) in the 32-month-old animals. Neuronal lipofuscin accumulation in the neurons of cerebral motor cortex, cerebellum and cervical spinal cord was increased (P < 0.05) in the older animals. Oxygen consumption was found to be decreased significantly(P < 0.05) in the 32-month old guinea pigs. Treatment with MAK at a dose of 500 mg/kg body weight daily for two months reduced the lipid peroxidation and lipofuscin pigment accumulation significantly in brain regions and it also helped in restoring the normal oxygen consumption in the older animals. This indicates antioxidant properties of MAK.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

Correlation of isonicotinic acid hydrazide infiltration across the blood-brain barrier to postnatal development in rats.

Changes in the isonicotinic acid hydrazide (INH) concentration in rat blood and brain were studied in correlation to postnatal development in groups of animals aged 21 and 42 days and 3 months. In the first part of the experiments, INH was administered intravenously to all the age groups in a dose of 100 mg/kg. In the second part, the dose was related to extracellular fluid volume, so that the 3-week-old rats were given 154 mg/kg, the 6-week-old animals 129 mg/kg and the 3-month-old animals 100 mg/kg. After a dose of 100 mg/kg, INH levels in the blood of 21-day-old rats were significantly lower than in 42-day-old and adult animals. The brain INH levels did not differ significantly. On relating the dose to the amount of extracellular fluid, there were no significant differences in the blood INH levels, but the levels in the brain of 21- and 42-day-old rats were significantly higher than in 3-month-old animals. Blood volume related to body weight and brain weight did not differ in the various age groups. The authors conclude that the blood-brain barrier for isonicotinic acid hydrazide alters in rats during postnatal development. In young animals (21- and 42-day-old), more INH infiltrates into the CNS than in adult animals.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Differential Changes in Superoxide Dismutase Activity in Brain and Liver of Old Rats and Mice

Superoxide dismutase (SOD) activity was measured in the brain and liver of 24–26- and 3-month-old rats. No significant age-related differences in Cu/Zn-SOD activity were found in any of the tissues studied. A small but significant increase in total SOD activity was observed in the whole brain (10-20%), cerebral cortex (11%), and hypothalamus (18%) of old rats, whereas a much more important increase in Mn-SOD activity was found in the whole brain (48%), cerebral cortex (70%), striatum (60%), and hypothalamus (30%). The increase of Mn-SOD activity in the brain of old rats suggests the enzyme may play an important role in the process of aging. Mn-SOD is found only in the mitochondrion, which could be an important site of oxygen free radical production, and a significant increase in the enzyme activity was also found in the lung of hypoxic rats. A significant decrease in total SOD and Mn-SOD activity was observed in the liver of old rats. Preliminary experiments in 23–24-month-old mice similarly showed an increase and a decrease in total SOD and Mn-SOD activity, respectively, in the whole brain and liver. These results suggest that the regulatory mechanisms of Mn-SOD in the brain and liver vary differentially with age.     

Duodenal endocrine cells in mice with particular regard to age-induced changes

Duodenal endocrine cell types in four age groups of NMRI mice (1, 3, 12 and 24 months old) were identified by immunocytochemistry and quantified by computerized image analysis. Whereas the number of secretin-immunoreactive cells was significantly increased in the 24-month-old group, the number of GIP-immunoreactive cells was reduced in 12-month-old compared with 3-month-old mice. The number of somatostatin-immunoreactive cells was fewer in both the 12- and 24-month-olds vis-à-vis the 3-month-old mice. Whereas serotonin-immunoreactive cells were fewer in both 1-month-old and 12-month-old mice, they were more numerous in 24-month-old mice then in the 3-month-old ones. The number of gastrin/CCK-immunoreactive cells was unaffected by age. The cell secretory index (CSI) of secretin- and serotonin-immunoreactive cells was increased in the 24-month-old mice vis-à-vis the 3-month-old ones and the CSI of GIP- and somatostatin-immunoreactive cells was increased in 12-month-old mice vis-à-vis 3-month-old mice. In contrast, the CSI of somatostatin- and serotonin-immunoreactive cells in 1-month-old mice was lower than that of 3-month-old-mice. The nuclear volume of secretin-, GIP-, gastrin/CCK- and serotonin-immunoreactive cells was less in 1-month-olds than in 3-month-old mice. Whereas the nuclear volume of somatostatin-immunoreactive cells was decreased in 12-month-old animals, that of gastrin/CCK- and serotonin-immunoreactive cells was greater in 24-month-old mice than in 3-month-old ones. It is concluded that these changes may be secondary to structural and functional changes in the gastrointestinal tract caused by ageing. It is possible that these changes are involved in the development of dysfunction of the gut observed at advanced age.     

Experimental model of brain amyloidosis in old rats

An experimental model of brain amyloidosis, based on the widely known casein model, has been developed. In adult 8–10-month-old rats, the latter model reproduces amyloidosis of internal organs, but does not involve the brain. At the same time, in old (26–28-month-old) rats injection of sodium caseinate results in accumulations of amyloid not only in the vascular walls of the kidneys, heart, lien, and liver, but also in the walls of pial vessels and radial cortical arteries and, in more rare cases, in the walls of large vessels in the brain base. Together with the structural impairments, injections of sodium caseinate evoke considerable shifts of the parameters of background mass electrical activity in the basolateral amygdala, dorsal hippocampus, and parietal cortex. Relative content of slow and sometimes activity increased, while the share of high-frequency waves dropped. These changes were relatively moderate in adult rats, while they were much more pronounced in old rats. Investigation of behavioral phenomena showed that after injections of sodium caseinate the ability to memorize becomes suppressed, and manifestations of the depressive state are intensified. Combined injections of sodium caseinate and phenobollne, the stimulant of protein synthesis, resulted in the development of more intensive morphological and functional manifestations of amyloidosis in old rats.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 83–94, March–June, 1996.