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.     

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.     

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.     

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.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

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.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Expression and Changes of Hyperoxidized Peroxiredoxins in Non-Pyramidal and Polymorphic Cells in the Gerbil Hippocampus During Normal Aging

Oxidative stress is one of predisposing factors to age-related neurodegeneration in the brain. In particular, thiol-containing groups are susceptible to oxidative stress, which induces the formation of the disulfide bond and/or hyperoxidized form of thiol-containing proteins. We observed the protein thiol levels in the hippocampal homogenates and also investigated changes in hyperoxidized form of peroxiredoxin (Prx–SO₃) immunoreactivity and proteins levels in the gerbil hippocampal subregions during normal aging. Levels of total thiol, non-protein thiol, and protein thiol were decreased in the hippocampal homogenates with age. At post-natal month 1 (PM 1), pyramidal and non-pyramidal cells in the hippocampal CA1 region (CA1) showed Prx–SO₃ immunoreactivity. Prx–SO₃ immunoreactivity in the cells was decreased by PM 12, thereafter, Prx–SO₃ immunoreactivity in the cells increased again with age. In the CA2/3, Prx–SO₃ immunoreactivity in pyramidal cells was not significantly changed; however, the immunoreactivity in pyramidal cells was very low at PM 12. Prx–SO₃ immunoreactivity in the dentate gyrus (DG) was distinctly changed during aging. At PM 1, Prx–SO₃ immunoreactivity in granule and polymorphic cells was weak and strong, respectively. The immunoreactivity in the neurons was decreased with age, not shown in any neurons at PM 12. Thereafter, Prx–SO₃ immunoreactivity increased again with age. In addition, Prx–SO₃ protein level in the hippocampus was lowest at PM 12. These results suggest that thiol-containing proteins are changed during aging and Prx–SO₃ immunoreactivity was different according to cells in the hippocampal subregion during aging.     

Comparison of Phosphorylated Extracellular Signal-Regulated Kinase 1/2 Immunoreactivity in the Hippocampal Ca1 Region Induced by Transient Cerebral Ischemia Between Adult and Aged Gerbils

In this study, the authors examined the difference of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the hippocampal CA1 region (CA1) between adult and aged gerbils after 5 min of transient cerebral ischemia. Delayed neuronal death in the CA1 of the aged group was much slower than that in the adult group after ischemia/reperfusion (I/R). pERK1/2 immunoreaction was observed in the CA1 region of the sham-operated adult gerbil. pERK1/2 immunoreactivity and protein levels in the ischemic CA1 region of the adult group were markedly increased 4 days after I/R, and then reduced up to 10 days after I/R. In contrast, pERK1/2 immunoreaction was hardly detected in the CA1 region of sham-operated aged gerbils, and the immunoreactivity increased from 1 day after the ischemic insult, and still observed until 10 days post-ischemia. In addition, pERK1/2-immunoreaction was expressed in astrocytes in the ischemic CA1 region: The expression in the ischemia-operated aged gerbils was later than that in the ischemia-operated adult gerbils. These results indicate that different patterns of ERK1/2 immunoreactivity may be associated with different processes of delayed neuronal death in adult and aged animals.     

Decreased Glucagon-Like Peptide-1 Receptor Immunoreactivity in the Dentate Granule Cell Layer from Adult in the Gerbil Hippocampus

In this study, we investigated age-related changes in glucagon-like peptide-1 receptor (GLP-1R) immunoreactivity and its protein levels in the gerbil hippocampus during normal aging. In the postnatal month 3 (PM 3) group, GLP-1R immunoreaction was well observed in neurons, especially pyramidal and non-pyramidal cells in the hippocampus proper, and granule and polymorphic cells in the dentate gyrus. In the hippocampus proper, GLP-1R immunoreactivity in neurons was maintained until PM 24. In the dentate gyrus, however, GLP-1R immunoreactivity in granule cells, not polymorphic cells, was hardly detected from PM 6. Western blot analysis also showed that age-dependent change patterns in GLP-1R protein levels in the gerbil hippocampus were similar to the immunohistochemical changes. These results indicate that GLP-1R immunoreactivity was markedly decreased in dentate granule cells from PM 6, showing that GLP-1R immunoreactivity and its protein levels were decreased in the adult and aged gerbil hippocampus.     

Age-related Changes in the Insulin Receptor β in the Gerbil Hippocampus

The insulin receptor has been reported to be associated with memory formation via the hippocampus. In this study, we observed age-related changes in the insulin receptor β immunoreactivity and its protein levels in the hippocampus of gerbils of various ages in order to identify the correlation between the insulin receptor β and aging processes in the hippocampus. Insulin receptor β immunoreactivity was mainly detected in the molecular and polymorphic layers of the dentate gyrus, and in mossy fibers, Schaffer collaterals, alveus and stratum lacunosum-moleculare of the hippocampus proper (CA1-3) of gerbils at postnatal month 1 (PM 1). Insulin receptor β immunoreactivity decreased with age in all of these structures, except for the alveus. Reduction of the insulin receptor β immunoreactivity was prominent in the molecular layer of the dentate gyrus at PM 6 and in the stratum lacunosum-moleculare of the CA1 region at PM 12, while insulin receptor β immunoreactivity was decreased in other regions in the PM 18 groups. In addition, insulin receptor β protein level in the whole hippocampus was slightly increased at PM 3, and it decreased in an age-dependent manner from PM 6 to PM 24. These reductions of the insulin receptor β in the hippocampus may be associated with age-related memory deficits in gerbils.     

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.     

Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult

In the present study, we focused upon expression and changes of endogenous insulin-like growth factor-1 (IGF-1) in the hippocampus of the Mongolian gerbil after ischemic insult. In sham-operated animals, IGF-1 immunoreactivity was absent from the hippocampus. IGF-1-immunoreactive (IR) neurons were detected at 12 h and 1 day after ischemic insult. In the hippocampal CA1 area, the IGF-IR neurons were non-pyramidal cells (GABAergic neurons). In the hippocampal CA2/3 areas, the IGF-1-IR neurons were pyramidal and non-pyramidal cells, and in the dentate gyrus the IGF-1-IR neurons were hilar neurons. Four days after ischemia-reperfusion, IGF-1 immunoreactivity disappeared from neurons, and significantly increased in astrocytes and microglia. These results suggest that the induction of IGF-1 in the CA1 area during the early stage (12-24 h after ischemic insult) is associated with the relative vulnerabilities of pyramidal glutamatergic neurons and non-pyramidal GABAergic neurons. The later increase (4 days after ischemic insult) of IGF-1 expression and protein content was found to promote the activities of astrocytes and microglia. These increases of IGF-1 in astrocytes and in microglia are associated with mechanisms that compensate for the effects of delayed neuronal death.     

Comparison of Glial Activation in the Hippocampal CA1 Region Between The Young and Adult Gerbils After Transient Cerebral Ischemia

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5?min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4?days posti-schemia; the neuronal death in the young gerbil occurred at 7?days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.     

Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia

We investigated changes of immunoreactivities of N-methyl-D-aspartate receptor (NR) and of excitatory amino acid carrier 1 (EAAC-1), the neuronal glutamate transporter, in the vulnerable CA1 area and the less vulnerable subiculum of the gerbil hippocampus at various times following transient forebrain ischemia. At 30 min after ischemia-reperfusion, the intensity of NR immunoreactivity increased markedly in neurons of CA1 and subiculum, particularly NR2A/B, while EAAC-1 immunoreactivity was reduced in CA1. At 3 hr after reperfusion, the density of NR1 immunoreactivity markedly decreased in CA1. In contrast EAAC-1 immunoreactivity increased in CA1 and in the subiculum. At 12 hr after reperfusion, the decrease of NR1 immunoreactivity was not detected whereas EAAC-1 immunoreactivities in the CA1 area were intensified. In the subiculum, both NR subunits immunoreactivities decreased significantly, in contrast to the maintenance of EAAC-1 immunoreactivity. At 24 hr after reperfusion, both NR2A/B and EAAC-1 immunoreactivities decreased markedly in CA1 and subiculum. We tentatively suggest that the increase of NR immunoreactivity in CA1 at early times after ischemia-reperfusion may increase the delayed neuronal death, and that the increase or maintenance of EAAC-1 immunoreactivity at early times after ischemia-reperfusion may be an important factor in survival of neurons.     

Time Course of Changes in Immunoreactivities of GABA Degradation Enzymes in the Hippocampal CA1 Region after Adrenalectomy in Gerbils

Adrenalectomy (ADX) has been useful for a good in vivo model for apoptosis in the hippocampus by the absence of corticosteroids following ADX. In some neurodegenerative diseases, GABAergic neurons are more resistant to neuronal damage as compared with glutamatergic neurons. In the present study, we observed chronological changes in three GABA degradation enzymes, e.g., GABA transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivity and protein levels in the gerbil hippocampal CA1 region after ADX. Changes in their immunoreactivities were distinct in the stratum pyramidale of the CA1 region. GABA-T immunoreactivity and protein level were significantly increased in the CA1 region 3 h after ADX, in contrast, SSAR and SSADH immunoreactivity and protein level were increased 12 h and 3–12 h, respectively, after ADX. These results suggest that the increases of GABA-T, SSADH and SSAR immunoreactivity and protein levels in the hippocampal CA1 region in ADX gerbils may be associated with the control of GABA levels in this region.     

Microtubule-Associated Protein 2 as an Early Indicator of Ischemia-Induced Neurodegeneration in the Gerbil Forebrain

Abstract: Microtubule-associated protein 2 (MAP-2) was studied in the gerbil hippocampus and striatum after transient ischemia. Western immunoblot analysis shows that there is a significant decrease of MAP-2 in the dorsolateral sector of the striatum and a slight decrease of MAP-2 in the CA1 region of the hippocampus 6–12 h after ischemia in the gerbil forebrain. The immunohistochemical staining pattern of MAP-2 in these two regions also shows a loss of immunostaining of MAP-2. In particular, a beaded MAP-2 immunostaining pattern at the apical dendritic region of the CA1 neurons of the hippocampus was found within 12 h after ischemia compared with the smooth dendritic immunostaining of MAP-2 in normal CA1 neurons. In vitro assays of MAP-2 degradation suggest that dendritic loss of immunoreactivity after ischemia seen on western blots may be due to calpain I degradation of MAP-2. Loss of MAP-2 in both the striatum and hippocampus was found to occur earlier than spectrin degradation by western blot analysis. These results suggest that loss of MAP-2 may participate in the initial phase of neuronal dysfunction and that dendritic breakdown may be a first sign of neurodegeneration.     

Age-related Differentiation in Newly Generated DCX Immunoreactive Neurons in the Subgranular Zone of the Gerbil Dentate Gyrus

In the present study, we investigated age-related changes of newborn neurons in the gerbil dentate gyrus using doublecortin (DCX), a marker of neuronal progenitors which differentiate into neurons in the brain. In the postnatal month 1 (PM 1) group, DCX immunoreactivity was detected in the subgranular zone of the dentate gyrus, but DCX immunoreactive neurons did not have fully developed processes. Thereafter, DCX immunoreactivity and its protein levels in the dentate gyrus were found to decrease with age. Between PM 3 and PM 18, DCX immunoreactive neuronal progenitors showed well-developed processes which projected to the granular layer of the dentate gyrus, but at PM 24, a few DCX immunoreactive neuronal progenitors were detected in the subgranular zone of the dentate gyrus. DCX protein level in the dentate gyrus at PM 1 was high, thereafter levels of DCX were decreased with time. The authors suggest that a decrease of DCX immunoreactivity and its protein level with age may be associated with aging processes in the hippocampal dentate gyrus.