Yoshihiro Kiyota

Age-related changes in learning and memory in the senescence-accelerated mouse (SAM)

Age-related changes in learning ability were studied in senescence-accelerated mice (SAM) reared under specific pathogen-free (SPF) conditions. SAM-P/8/Ta (SAM-P/8, senescence-prone substrain) showed an age-associated increase in spontaneous motor activity (SMA) compared with SAM-R/1/Ta (SAM-R/1, senescence-resistant substrain) in a novel environment when the activity was measured in the light period, although there was no significant difference in the dark period. In observations of the circadian rhythm of SMA, SAM-P/8 showed a significant increase in diurnal SMA. In SAM-P/8 mice, the acquisition of passive avoidance response was slightly but significantly impaired even at 2 months of age, compared with SAM-R/1 control; the impairment became obvious with aging. In a one-way active avoidance task, SAM-P/8 did not show any impairment in the acquisition of avoidance response at 2 and 4 months of age. However, significant impairment was observed in SAM-P/8 at 12 months of age. The impairments of avoidance tasks were not due to a decrease in shock sensitivity, as indicated by no significant change in the flinch-jump threshold. In a water-filled multiple T-maze task, there was no difference in the number of errors between the two groups. With regard to the performance time to reach the goal, however, SAM-P/8 showed a mild prolongation at 2 months of age, and the prolongation became marked with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between brain damage and memory impairment in rats exposed to transient forebrain ischemia

The relationship between changes in learning behavior and neurological damage following transient forebrain ischemia was studied in rats. The transient forebrain ischemia was induced by 4-vessel occlusion, and behavioral experiments were started 4 weeks later when histological damage to the brain seemed to have stabilized. Histological evaluation of brain damage was conducted after completion of the behavioral studies. The rats showed marked learning impairment in a radial maze task done from 4 to 10 weeks after ischemia. In particular, there was an increase in the number of working memory errors according to the duration of forebrain ischemia. However, the same rats showed good avoidance responses in a passive avoidance task done 12 weeks after ischemia. The rats also showed good acquisition of escape response in a water maze task carried out 13 weeks after ischemia, but showed slight impairment of spatial navigation in the transfer test. Marked neuronal degeneration was observed in the hippocampal pyramidal cells of the rats exposed to ischemia. This neuronal damage was closely related to memory impairment in the radial maze task, as demonstrated by a significant negative correlation (r = -0.609 or -0.709) between the number of surviving neurons and the number of reference or working memory errors. These results suggest that rats exposed to transient forebrain ischemia show marked impairment of both reference and working memories as a result of postischemic hippocampal damage.

Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia.

The amounts of mRNAs for proto-oncogene c-fos and structural protein beta-actin were measured in the rat cerebral cortex after transient forebrain ischemia. A transient and specific induction of c-fos mRNA was noticed in the cerebral cortex 30-90 min after ischemia followed by decline to control value. In contrast, the level of mRNA for beta-actin was not altered throughout the recirculation period examined. These results suggest specific role of c-fos gene after brain damage.

Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia

SummaryWe examined the time course of basic fibroblast growth factor (bFGF) immunoreactivity and its mRNA level mainly in the hippocampus after transient forebrain ischemia using immunohistochemistry, enzyme immunoassay (EIA), Western blot analysis and in situ hybridization. Neuronal death in the hippocampal CA1 subfield was observed 72 h after 20 min of ischemia. The number of bFGF-immunoreactive(IR) cells increased 48 h–5 days after ischemia in all hippocampal regions. At 10 and 30 days, the bFGF-IR cells in the CA1 subfield had further increased in numbers and altered their morphology, enlarging and turning into typical reactive astrocytes with the advancing neuronal death in that area. In contrast, the number of bFGF-IR cells in other hippocampal regions had decreased 30 days after ischemia. The EIA study showed a drastic increase in bFGF levels in the hippocampus 48 h after ischemia (150% of that in normal rat) which was followed by further increases. In Western blot analysis, three immunoreactive bands whose molecular weights correspond to 18, 22 and 24 kDa were observed in normal rat and ischemia increased all their immunoreactivities. In the in situ hybridization study of the hippocampus, bFGF mRNA positive cells were observed in the CA1 subfield in which many bFGF-IR cells existed after ischemia. These data demonstrate that transient forebrain ischemia leads to an early and strong induction of bFGF synthesis in astrocytes, suggesting that the role of bFGF is related to the function of the reactive astrocytes which appear following brain injury.

Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia

Hypothermia is the only neuroprotective therapy proven to be clinically effective. Identifying the molecules that play important roles in the efficacy of hypothermia, we developed a multi-channel computer-controlled system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 degrees C, and examined the time window necessary to exert its significant neuroprotective effects. Eight-week-old SD rats were subjected to a 2h middle cerebral artery occlusion (MCAO) with an intraluminal filament, and post-ischemic hypothermia was introduced at 0, 2, 4, or 6h after reperfusion until the rats were killed 2 days after MCAO. Since a significant protection was observed when hypothermia was started within 4h after reperfusion, it was concluded that the therapeutic time window of mild hypothermia lasts for 4h after reperfusion in our model. On the basis of the window, comprehensive gene expression analyses using oligonucleotide microarrays were conducted and identified potential genes related to the efficacy of hypothermia, which included inflammatory genes like osteopontin, early growth response-1, or macrophage inflammatory protein-3alpha. Therefore, the neuroprotective effects of post-ischemic mild hypothermia were strongly suggested to be mainly associated with the reduction of neuronal inflammation.

Senescence-accelerated mouse (SAM): Age-related reduced anxiety-like behavior in the SAM-P/8 strain

Age-related behavioral changes in the passive avoidance, food neophobia, elevated plus-maze, and water-lick conflict tests were studied using substrains of senescence-accelerated mouse (SAM-P/8 and SAM-R/1) at 2 to 20 months of age. SAM-P/8 mice exhibited a significant impairment of acquisition of passive avoidance compared with SAM-R/1 mice when they were trained repeatedly, and the acquired response in SAM-P/8 mice rapidly diminished in contrast to good retention in SAM-R/1 mice. SAM-P/8 mice showed an age-related decrease in the latency to eat novel food after a 24-h food deprivation as compared with SAM-R/1 mice at 2 to 12 months of age, despite no significant difference in latency to eat familiar food between the two strains. In the elevated plus-maze test, SAM-P/8 mice had apparent increases in the number of entries into open arms and time spent on open arms in comparison to SAM-R/1 mice at 4 through 12 months of age; this difference became obvious with aging, implying age-associated reduced anxiety in the SAM-P/8 strain. In addition, SAM-P/8 mice exhibited a significant increase in punished water drinking compared to SAM-R/1 mice in the water-lick conflict test, although unpunished water intake in SAM-P/8 mice did not differ from that in the SAM-R/1 control. Aged SAM-R/1 mice, 20 months old, exhibited low anxiety-like behavior in the food neophobia and elevated plus-maze tests such as was seen in SAM-P/8 mice, when compared with young (4-month-old) SAM-R/1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.

Macrophage inflammatory protein-3alpha plays a key role in the inflammatory cascade in rat focal cerebral ischemia.

Although hypothermia is one of the most robust neuroprotectants clinically available, its underlying mechanisms remain unclear. Through microarray gene expression analysis, we previously identified several key molecules potentially involved in the efficacy of hypothermia in a 2h middle cerebral artery occlusion (MCAO) rat model, including cytokine and chemokine genes. The present study demonstrated that the expressions of 2 genes, macrophage inflammatory protein-3alpha (MIP-3alpha) and its receptor, CC-chemokine receptor 6 (CCR6), were upregulated in the model and were suppressed by hypothermia. To investigate the role of cerebral MIP-3alpha, it was administered into the rat striatum; dose- and time-dependent induction of CCR6 gene expression was observed. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha injection also induced sequential expressions of MIP-3alpha and CCR6. MIP-3alpha was found to be produced by proinflammatory cytokines in rat astrocytes, while it was suppressed by hypothermia. In turn, MIP-3alpha stimulated IL-1beta and inducible nitric oxide synthase expressions in rat microglia and rat brains. Furthermore, intracerebroventricular administration of an anti-rat MIP-3alpha-neutralizing antibody significantly reduced the infarct in MCAO rat brains. These findings suggest that MIP-3alpha plays a pivotal role in inflammatory cascades in ischemic brains, and may be a novel therapeutic target for cerebral ischemia.

Hypothermia enhances heat-shock protein 70 production in ischemic brains.

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Cerebral embolization leads to memory impairment of several learning tasks in rats

The effects of cerebral embolization, produced by injecting microspheres into the left internal carotid artery, on passive and active avoidance tasks and water filled multiple T-maze task, were studied in male Wistar rats. The rats with cerebral embolization were markedly impaired acquisition and retention of the one-trial passive avoidance response. The impairment depended on the number of microspheres injected and continued for 2 weeks. The cerebral embolized rats were also impaired acquisition of two-way active avoidance response in a shuttle box. These impairments are not due to decrease in shock sensitivity, because there was no significant change in the flinch-jump threshold. The embolized rats also exhibited a significant disturbance in performance of water filled multiple T-maze learning. These results suggest that rats with cerebral embolization are impaired in three different types of learning tasks, and may be useful as an animal model for the vascular type of dementia.