Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

Chronological Distribution of Rip Immunoreactivity in the Gerbil Hippocampus During Normal Aging

Age-dependent studies on oligodendrocytes, which are the myelinating cells in the central nervous system, have been relatively less investigated. We examined age-dependent changes in Rip immunoreactivity and its protein level in the gerbil hippocampus during normal aging using immunohistochemistry and Western blot analysis with Rip antibody, an oligodendrocyte marker. Rip immunoreactivity and its protein level in the hippocampal CA1 region significantly increased at postnatal month 3 (PM 3). Thereafter, they decreased in the hippocampal CA1 region with age. At PM 24, Rip immunoreactive processes in the hippocampal CA1 region markedly decreased in the stratum radiatum. In the hippocampal CA2/3 region and dentate gyrus, the pattern of changes in Rip immunoreactivity and its protein level was similar to those in the hippocampal CA1 region; however, no significant changes were found in the CA2/3 region and dentate gyrus at various age stages. These results indicate that Rip immunoreactivity and protein level in the hippocampal CA1 region decreases significantly at PM 24 compared to the CA2/3 region and dentate gyrus.     

Parvalbumin Immunoreactivity and Protein Level are Altered in the Gerbil Hippocampus During Normal Aging

Hippocampal interneurons are local circuit neurons which are responsible for inhibitory activity in the hippocampus. Parvalbumin (PV) is one of useful markers for GABAergic interneurons, not for principle cells, in the hippocampus. In the present study, we investigated age-related changes in PV immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. PV immunoreactive neurons were detected in all hippocampal subregions of all groups. PV immunoreactive neurons, which innervated principal neurons, were non-pyramidal neurons in the hippocampal CA1-3 regions, and were polymorphic neurons in the dentate gyrus. In the hippocampal CA1 region, the number of PV immunoreactive neurons was significantly reduced in the postnatal month 3 (PM 3) group, which was sustained by PM 18, and, at PM 24, the number of PV immunoreactive neurons was significantly decreased. In the CA2/3 region and dentate gyrus, the number of PV immunoreactive neurons was significantly decreased at PM 6: Thereafter, the number of PV immunoreactive neurons was sustained until PM 24. In addition, changes in PV protein levels in the gerbil hippocampus were similar to immunohistochemical changes during normal aging: PV protein levels were significantly decreased with age by PM 6: Thereafter, PV protein levels were sustained by PM 24. These results suggest that PV immunoreactive interneurons were decreased in the hippocampus with age in gerbils.     

Regulation of Myelin Basic Protein Phosphorylation by Mitogen-Activated Protein Kinase During Increased Action Potential Firing in the Hippocampus

Myelin basic protein (MBP) phosphorylation is a complex regulatory process that modulates the contribution of MBP to the stability of the myelin sheath. Recent research has demonstrated the modulation of MBP phosphorylation by mitogen-activated protein kinase (MAPK) during myelinogenesis and in the demyelinating disease multiple sclerosis. Here we investigated the physiological regulation of MBP phosphorylation by MAPK during neuronal activity in the alveus, the myelinated output fibers of the hippocampus. Using a phosphospecific antibody that recognizes the predominant MAPK phosphorylation site in MBP, Thr95, we found that MBP phosphorylation is regulated by high-frequency stimulation but not low-frequency stimulation of the alveus. This change was blocked by application of tetrodotoxin, indicating that action potential propagation in axons is required. It is interesting that the change in MBP phosphorylation was attenuated by the reactive oxygen species scavengers superoxide dismutase and catalase and the nitric oxide synthase inhibitor N-nitro-L-arginine. Removal of extracellular calcium also blocked the changes in MBP phosphorylation. Thus, we propose that during periods of increased neuronal activity, calcium activates axonal nitric oxide synthase, which generates the intercellular messengers nitric oxide and superoxide and regulates the phosphorylation state of MBP by MAPK.     

Cyclooxygenase-2 Immunoreactivity and Protein Level in the Gerbil Hippocampus During Normal Aging

Cyclooxygenases-2 (COX-2) is not only related to inflammation but also plays critical roles in brain development and synaptic signaling. In the present study, we investigated age-related changes in COX-2 immunoreactivity and protein levels in the gerbil hippocampus. In the hippocampal CA1 region (CA1) and dentate gyrus (DG), weak COX-2 immunoreactivity was observed at postnatal month 1 (PM 1), and COX-2 immunoreactivity was markedly increased at PM 18 and 24. In the CA2/3, COX-2 immunoreactivity was strong at PM 1. COX-2 immunoreactivities in the PM 3, 6 and 12 groups were decreased compared to that in the PM 1 group, and it was increased at PM 18 and 24. In addition, age-related changes in COX-2 levels were similar with immunohistochemical results in the CA2/3. These results suggest that COX-2 immunoreactivity and levels were high in the hippocampus of aged gerbils.     

Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Calbindin D-28k Immunoreactivity and Its Protein Level in Hippocampal Subregions During Normal Aging in Gerbils

The hippocampus is associated with learning and memory function and shows neurochemical changes in aging processes. Calbindin D-28k (CB) binds calcium ion with a fast association rate. We examined age-related changes in CB immunoreactivity and its protein level in the gerbil hippocampus during normal aging. In the hippocampal CA1 region (CA1) and CA2, CB immunoreaction was found in some neurons in the stratum pyramidale (SP) at postnatal month 1 (PM 1). CB immunoreactivity in neurons was markedly increased at PM 3. Thereafter, CB immunoreactivity was decreased with time: CB-immunoreactive (⁺) neurons were fewest at PM 24. In the CA3, a few CB⁺ neurons were found only in the SP at PM 1 and in the stratum radiatum at PM 18 and 24. In addition, mossy fibers were stained with CB at PM 1. CB immunoreactivity in mossy fibers was markedly increased at PM 3, thereafter it was decreased with time. In the dentate gyrus, many granule cells (GC) in the granule cell layer were stained with CB at PM 1. CB immunoreactivity in GC was markedly increased at PM 3, thereafter CB immunoreactivity was decreased with time. In Western blot analysis, CB protein level in the gerbil hippocampus was highest at PM 3, thereafter CB protein levels were decreased with time. This result indicates that CB in the gerbil hippocampus is abundant at PM 3 and is decreased with age.     

Changes of Free Long-Chain Bases in Neuronal Cells During Differentiation and Aging in Culture

Abstract: Changes in the free long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o -phthalaldehyde and fractionated by reversed-phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: C18-sphingosine, C18-sphinganine, C20-sphingosine, and C20-sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18-sphingosine> C20-sphinganine and varying from 0.02 ± 0.015 pmol/mg of cell protein for C20-sphinganine at day 0 to 223 ± 22 pmol/mg of cell protein for C18-sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20-sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20-sphinganine progressively increased from day 0 to 22 of culture; that of C18-sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18- and C20-sphingosine levels during cell culture were qualitatively similar to those of C18- and C20-sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.     

Induction of Transcription Factor A-myb Expression in Reactive Astrocytes Following an Excitotoxic Lesion in the Mouse Hippocampus

In the present study, we examined patterns of A-myb expression in the kainic acid (KA)-treated mouse hippocampus. Western blot analysis revealed that A-myb expression was dramatically increased in brain 3 days after KA treatment, and was sustained for more than 7 days. A-myb immunoreactivity was restricted to hippocampal neurons in control mice. Three days after KA treatment, strong A-myb immunoreactivity was observed in reactive astrocytes throughout the CA3 region. Thereafter, A-myb immunoreactive astrocytes gradually concentrated around the CA3 region in parallel with selective neuronal loss, and only a few A-myb immunoreactive astrocytes persisted in the CA3 region 14 days after KA treatment. These findings suggest that the A-myb plays a role in the reactive gliosis signaling pathway in KA-induced excitotoxic lesions.     

Changes in the Ganglioside Long-Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Abstract: Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Transplantation of Foetal Neural Stem Cells into the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair. Maria Concetta Geloso and Stefano Giannetti contributed equally to this work.     

Age-associated cellular relocation of Sod 1 as a self-defense is a futile mechanism to prevent vascular aging

Vascular aging is characterized by the presence of chronic oxidative stress. Although cytosolic Sod 1 has a key role in the detoxification of superoxide ((*)O(2)(-)), little is known about its importance in vascular aging. We found that inhibition of Sod 1 had no effect on (*)O2- generation. Furthermore, its expression decreased in an age-dependent manner. Interestingly, Sod 1 loses its membrane-association and is also lost from the caveolae with increasing age. Instead, a relocation of Sod 1 to the mitochondria takes place, presumably in an attempt to maintain mitochondrial integrity and to counter-balance age-associated oxidative stress. Unlike Sod 2, which is constitutively expressed in mitochondria to control (*)O2- radical fluxes, Sod 1 is not inactivated by peroxynitrite and is not nitrated as a function of age. These novel insights into oxidative stress-associated vascular aging and the understanding about how redox-systems are regulated in old age may identify new targets to ameliorate aging as the greatest cardiovascular risk factor.     

Oxidation of KCNB1 K~+ channels in central nervous system and beyond

KCNB1, a voltage-gated potassium(K+) channel that conducts a major delayed rectifier current in the brain, pancreas and cardiovascular system is a key player in apoptotic programs associated with oxidative stress. As a result, this protein represents a bona fide drug target for limiting the toxic effects of oxygen radicals. Until recently the consensus view was that reactive oxygen species trigger a pro-apoptotic surge in KCNB1 current via phosphorylation and SNARE-dependent incorpora-tion of KCNB1 channels into the plasma membrane. However, new evidence shows that KCNB1 can be modified by oxidants and that oxidized KCNB1 channels can directly activate pro-apoptotic signaling pathways. Hence, a more articulated picture of the pro-apoptotic role of KCNB1 is emerging in which the protein induces cell's death through distinct molecular mechanisms and activation of multiple pathways. In this review article we discuss the diverse functional, toxic and protective roles that KCNB1 channels play in the major organs where they are expressed.     

Age-Dependent Changes in Calretinin Immunoreactivity and its Protein Level in the Gerbil Hippocampus

Calretinin (CR)-immunoreactive interneurons are well known as the interneuron specific interneurons in the hippocampus. CR-immunoreactive neurons form cellular network and regulate the activity of other GABAergic inhibitory interneurons in the hippocampus. In the present study, we investigated age-related changes in CR-immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. In all subregions of the gerbil hippocampus, the number of CR-immunoreactive neurons was significantly decreased in the postnatal month 6 (PM 6) group compared to that in the PM 1 group. Thereafter, CR-immunoreactive neurons were decreased with age. In addition, the number of CR-immunoreactive cells in the subgranular zone were significantly decreased in the PM 6 group. We also observed that CR protein levels were decreased gradually with age. These results indicate that both CR immunoreactivity and its protein level were decreased with age in the gerbil hippocampus during normal aging.     

Caenorhabditis elegans as a screening tool for the endothelial cell-derived putative aging-related proteins detected by proteomic analysis

Endothelial cells go through progressive pathophysiologic modification as cellular senescence progresses. In vitro, endothelial cell senescence is accompanied by failure of proliferation and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in the organism in vivo and free radical theory is accepted to be the most plausible explanation for this process. We have screened proteins involved in both cellular senescence and reactive oxygen species induced condition using 2-D gel analysis and found that ubiquitin carboxyl terminal hydrolase L1, peroxyredoxin 2, peroxyredoxin 4, fatty acid binding proteins (FABPs), and 5'-AMP-activated protein kinase beta-1 subunit were candidate aging-related proteins. To evaluate in vivo function of these proteins, Caenorhabditis elegans (C. elegans) knock-down system using RNA interference was applied. Aging-specific expression of lipofucsin and the lifespan of knocked-down C. elegans were observed to assess the outcome. Interestingly, the inhibition of the genes led to short lifespan and earlier accumulation of lipofucsin with increasing age when compared with the wild type. These results suggest that the above genes may be related to cellular senescence process in determining the longevity in C. elegans and that gene inactivation renders animals susceptible to oxidative stress.     

Metabolic Changes in Cerebral Cortex, Hippocampus, and Cerebellum During Sustained Bicuculline-Induced Seizures

Abstract— The objective of the present experiments was to study metabolic correlates to the localization of neuronal lesions during sustained seizures. To that end, status epilepticus was induced by i.v. administration of bicuculline in immobilized and artificially ventilated rats, since this model is known to cause neuronal cell damage in cerebral cortex and hippocampus but not in the cerebellum. After 20 or 120 min of continuous seizure activity, brain tissue was frozen in situ through the skull bone, and samples of cerebral cortex, hippocampus, and cerebellum were collected for analysis of glycolytic metabolites, phosphocreatine (PCr), ATP, ADP, AMP, and cyclic nucleotides. After 20 min of seizure activity, the two “vulnerable” structures (cerebral cortex and hippocampus) and the “resistant” one (cerebellum) showed similar changes in cerebral metabolic state, characterized by decreased tissue concentrations of PCr, ATP, and glycogen, and increased lactate concentrations and lactate/ pyruvate ratios. In all structures, though, the adenylate energy charge remained close to control. At the end of a 2-h period of status epilepticus, a clear deterioration of the energy state was observed in the cerebral cortex and the hippocampus, but not in the cerebellum. The reduction in adenylate energy charge in the cortex and hippocampus was associated with a seemingly paradoxical decrease in tissue lactate levels and with failure of glycogen resynthesis (cerebral cortex). Experiments with infusion of glucose during the second hour of a 2-h period of status epilepticus verified that the deterioration of tissue energy state was partly due to reduced substrate supply; however, even in animals with adequate tissue glucose concentrations, the energy charge of the two structures was significantly lowered. The cyclic nucleotides (cAMP and cGMP) behaved differently. Thus, whereas cAMP concentrations were either close to control (hippocampus and cerebellum) or moderately increased (cerebral cortex), the cGMP concentrations remained markedly elevated throughout the seizure period, the largest change being observed in the cerebellum. It is concluded that although the localization of neuronal damage and perturbation of cerebral energy state seem to correlate, the results cannot be taken as. evidence that cellular energy failure is the cause of the damage. Thus, it appears equally probable that the pathologically enhanced neuronal activity (and metabolic rate) underlies both the cell damage and the perturbed metabolic state. The observed changes in cyclic nucleotides do not appear to bear a causal relationship to the mechanisms of damage.     

慢性避水应激大鼠肠道菌群和ROS的变化

目的探讨应激诱导的内脏高敏感大鼠中肠道菌群及活性氧簇(ROS)的变化。方法建立慢性避水应激大鼠模型,分为应激组和对照组(每组6只)。腹部回撤反射(abdominal withdrawal reflex,AWR)方法评估大鼠内脏敏感性。免疫荧光和ELISA方法检测肠道和血液中ROS的表达水平。粪便进行16S rDNA菌群测序分析。细胞培养方法检测大鼠结肠黏膜菌群代谢产物对巨噬细胞ROS生成的影响。结果慢性避水应激能诱导大鼠内脏敏感性增高。与对照组相比,应激组大鼠肠道和血液中ROS表达均增加,不同水平上肠道菌群都有所变化,生物多样性下降。Spirochaetia菌的丰富度与ROS的表达呈负相关。应激组大鼠结肠黏膜菌群诱导巨噬细胞产生更高水平的ROS。结论应激诱导大鼠肠道菌群发生紊乱引起ROS生成增加并存在相关性。