Kazuhiro Terai

β-amyloid deposits in transgenic mice expressing human β-amyloid precursor protein have the same characteristics as those in Alzheimer's disease

Abstract A transgenic mouse expressing the human β-amyloid precursor protein with the “Swedish” mutation, Tg2576, was used to investigate the mechanism of amyloid-β peptide (Aβ) deposition. We characterized Aβ deposits in the cerebral cortex biochemically and pathologically. A surface-enhanced laser desorption/ionization affinity mass spectrometric study using the 6E10 monoclonal antibody demonstrated that the major species of Aβ in a formic acid-extracted fraction of the cortex were Aβ 1–38 , Aβ 1–40 and Aβ 1–42 . Immunohistochemistry using antibodies to the carboxy-terminal epitopes of Aβ 1–40 and Aβ 1–42 , as well as 6E10, showed that plaques containing Aβ 1–42 were more numerous than those containing Aβ 1–40 throughout the cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that Aβ 1–40 was preferentially located in the central part of the Aβ 1–42 positive plaques. Enzyme-linked immunosorbent assay measurements of Aβ 1–40 and Aβ 1–42 showed that Aβ 1–40 was several-fold more abundant than Aβ 1–42 . From these data we suggest that Aβ 1–42 deposition may precede Aβ 1–40 deposition, while Aβ 1–40 begins to deposit in the central part of the plaques and accumulates there. Furthermore, localization of Aβ 1–40 corresponded almost exactly to congophilic structures, which were associated with aberrant swollen synapses detected with antibodies to synaptophysin and α-synuclein. Thus, Aβ deposits in Tg2576 mice have similar characteristics to those in Alzheimers disease.

Kainic acid-induced activation of nuclear factor-κB in rat hippocampus

Abstract Nuclear factor-κB (NF-κB) is known to play a key role in immune and inflammatory responses. To understand the mechanisms of inflammatory activation that accompany neuronal damage, we determined the cell type in which NF-κB was activated. NF-κB protein was detected in the cytosolic fraction of untreated and vehicle-treated rat hippocampus. After kainic acid (KA) treatment, NF-κB protein was significantly increased in both the cytosolic and particulate fractions. NF-κB immunoreactivity was observed in both brain blood vessels and glial cells after 1 day. Although NF-κB immunoreactivity in brain blood vessels disappeared after 3 days, this activity was maintained in glial cells for up to 7 days. In addition, double immunostaining indicates that NF-κB was activated in glial cells, such as microglia and astrocytes, after 3 days. Thus, NF-κB activation seems to be delayed and to occur continuously in microglia and astrocytes, suggesting that an inflammatory activation in glial cells participates in KA-induced neurodegeneration.

Amount of Bleeding and Hematoma Size in the Collagenase-Induced Intracerebral Hemorrhage Rat Model

The aggravated risk on intracerebral hemorrhage (ICH) with drugs used for stroke patients should be estimated carefully. We therefore established sensitive quantification methods and provided a rat ICH model for detection of ICH deterioration. In ICH intrastriatally induced by 0.014-unit, 0.070-unit, and 0.350-unit collagenase, the amount of bleeding was measured using a hemoglobin assay developed in the present study and was compared with the morphologically determined hematoma volume. The blood amounts and hematoma volumes were significantly correlated, and the hematoma induced by 0.014-unit collagenase was adequate to detect ICH deterioration. In ICH induction using 0.014-unit collagenase, heparin enhanced the hematoma volume 3.4-fold over that seen in control ICH animals and the bleeding 7.6-fold. Data suggest that this sensitive hemoglobin assay is useful for ICH detection, and that a model with a small ICH induced with a low-dose collagenase should be used for evaluation of drugs that may affect ICH.

Immunohistochemical localization of GABAA receptors in comparison with GABA-immunoreactive structures in the nucleus tractus solitarii of the rat

The localization of GABAA receptors was studied by immunohistochemistry in the nucleus tractus solitarii of the rat using a monoclonal antibody (bd17) against the beta-subunit. The pattern of distribution was compared with that of GABA-immunoreactive axons and nerve terminals. Positive staining for GABAA receptors was confined to regions near the surface of neuronal somata and their processes. The highest density of positive staining for GABAA receptors was seen in the central part of the rostral nucleus tractus solitarii where GABA-positive terminals were also rather dense. At both intermediate and caudal levels of the nucleus tractus solitarii, a moderate density of positive staining for GABAA receptors was located in the ventrolateral part, including the ventrolateral subnucleus. In these regions, the density of GABA-positive terminals was low. In the medial nucleus tractus solitarii, including the medial subnucleus, very little or no positive staining for GABAA receptors was detected, although many GABA-positive terminals were observed. The results suggest that the central part of the rostral nucleus tractus solitarii is controlled by the GABAergic system via GABAA receptors, but in the medial subnucleus of the nucleus tractus solitarii the GABA neurons appear to act via receptors that are not detectable by the antibody used.

Delayed expression of c-fos protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia

The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O2 and 95% N2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.

Time course of in vitro expression of NADPH-diaphorase in cultured rat brain neurons : comparison with in vivo expression

The time course of NADPH-diaphorase expression was examined in primary cultures of rat central nervous system and in embryonic or neonatal rat brains using a histochemical method. In cerebral and brainstem cultures from 17-day-old embryonic rats, neuronal cells moderately expressing NADPH-diaphorase were first detected on about the 5th to 7th day of culturing. Both the density of positive cells and the staining intensity increased with age of cultures. The density of positive cells, calculated as a percent of the total number of cells, increased up to day 21 in cultures from both the cerebrum and the brainstem, indicating that NADPH-diaphorase is preferentially expressed in neurons with longer viability. On the other hand, virtually no intensely positive cells were detectable in cerebellar cultures at any period examined up to 21 days. In the in vivo study, moderately stained NADPH-diaphorase-positive neurons were first detected, mainly in the laterodorsal-pedunculopontine tegmental nuclei complex and partly in the striatum, in 16-day-old embryonic rat brain. At 2 days postnatal, intensely stained neurons were detectable in the cerebral cortex as well as in the tegmental nuclei complex and the striatum, indicating some delay in the in vitro, as compared to the in vivo, expression of neuronal NADPH-diaphorase.

Neuroprotective effects of NBQX on hypoxia-induced neuronal damage in rat hippocampus

Models of cerebral ischaemia were used for analysis of mechanism of neuronal cell death and/or damage. Ischaemia is caused dominantly by severe hypoxia and hypoglycaemia: in the present study, we examined the influence of severe in vivo hypoxia (5% O2/95% N2 for 30 min at 22 degrees C). After hypoxia, neuronal damage was observed in the CA3 and dentate gyrus (DG) after 3 and 21 days of survival, but not in the CA1.2,3-Dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), an antagonist for AMPA/kainate receptors, showed neuroprotective effects in the CA3 and DG. These results suggest that hypoxia may induce neuronal damage in the CA3 and DG through activation of AMPA/kainate receptors.

Effect of AMPA receptor antagonist YM872 on cerebral hematoma size and neurological recovery in the intracerebral hemorrhage rat model

[2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]-acetic acid monohydrate (YM872 or zonampanel), an AMPA receptor antagonist, is in clinical development for acute ischemic cerebral infarction. Stroke patients are prone to have subsequent intracerebral hemorrhages. In order to predict potential adverse effects, YM872 was tested in a rat model with collagenase-induced intracerebral hemorrhage. The morphologically determined hematoma volumes after 24 h were compared between animal groups intravenously infused with 3600 U/kg/h heparin for 30 min, or with 20 or 40 mg/kg/h of YM872, or placebo for 4 h. Heparin enlarged hematoma volume, but neither dose of YM872 affected hematoma size. In a separate study, neurological deficits were scored at various days after intracerebral hemorrhage induction in animals with intravenous infusion for 24 h of 10 or 20 mg/kg/h YM872, or saline. The YM872 groups scored significantly better than the saline group at 14 days. These data suggest that YM872 does not exacerbate intracerebral hemorrhage and might accelerate recovery.

Production of monoclonal antibody against histamine and its application to immunohistochemical study in the stomach.

A monoclonal antibody against histamine has been produced. A histamine–haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylami nopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or α-fluoromethylhistidine-treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mm histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with α-fluoromethylhistidine every 8 h. In these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach.