Masaya Nakashima

INVOLVEMENT OF FREE RADICALS IN CEREBRAL VASCULAR REPERFUSION INJURY EVALUATED IN A TRANSIENT FOCAL CEREBRAL ISCHEMIA MODEL OF RAT

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.

Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing

A novel tract-tracing procedure by using a sequential in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) is described. This method identifies fragmented DNA transported into neuronal fibers in tissue sections of gerbil hippocampal CA1 neurons following transient forebrain ischemia. The transported DNA has been confirmed by another method, fluorescence DNA detection technique by DAPI. Many methods have been developed to study the neuroanatomical connections in the central nervous system. Principally, these techniques are based on tract-tracing studies using xenobiotics into the central nervous system. Our tract-tracing method is originated from an intrinsic marker that is produced during the apoptotic process of neurons. Furthermore, the advantage of this method is that only the selected cells undergoing apoptosis are recognized and traced to the end of the related neuronal fiber. Usually, apoptotic cells possess intact intracellular metabolic mechanisms until completion of cell death. Thus, apoptotic neurons retain the axonal transport mechanisms which enables us to detect fragmented DNA moving from nuclei to distal terminals of neuronal fibers. Since TUNEL-positive DNA movement within neuronal fibers occurs only during a limited period, it is essential that a time-course of the TUNEL technique is used to study tract-tracing of apoptotic neurons. Although this method can identify only the apical dendrites of cells that are undergoing apoptosis during the limited period, some projections of the gerbil hippocampal CA1 neurons undergoing apoptosis are clearly demonstrated.

Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia.

The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial cells in experimental models of adult stroke. It has been reported that activated microglial cells are widely observed in the brain, including in the hippocampal CA1 region after transient ischemic insult. In the present study, time course expression of galectin-3 following transient forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1 immunostaining (a specific microglial cell marker), hematoxylin and eosin staining (for morphological observation), and in situ terminal dUTP-biotin nick end labeling of DNA fragments method (for determination of cell death). Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself. Our results indicate that galectin-3 might exert its effect by modulating the neuronal damage in delayed neuronal death.

Protective Effect of Apoptosis-Inhibitory Agent, N-Tosyl-l-Phenylalanyl Chloromethyl Ketone against Ischemia-Induced Hippocampal Neuronal Damage:

Delayed neuronal death in the gerbil hippocampal CA1 sector occurs 48 to 72 hours after severe forebrain ischemia. DNA fragmentation is observed in the hippocampal CA1 neurons at around that time. We show here that an inhibitor of proteolytic process of apoptosis, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), protected hippocampal neuronal damage by inhibition of the DNA fragmentation in a dose-dependent manner and that TPCK induced an apoptosis-regulating molecule, Bcl-2 protein, in the surviving neurons. These results suggest the prevention of apoptosis-related DNA fragmentation by TPCK may be an attractive therapeutic strategy for preserving hippocampal neurons from ischemic insult.

Relationship between magnitude of hypothermia during ischemia and preventive effect against post-ischemic DNA fragmentation in the gerbil hippocampus

Protective effect of hypothermia against DNA fragmentation in hippocampal CA1 field after transient forebrain ischemia in gerbils was evaluated by changing the magnitude of hypothermia. Inhibition of DNA fragmentation was proportional to the magnitude of hypothermia. The result indicates that, in terms of susceptibility to ischemia, hippocampal CA1 neurons are sensitive to a relatively small decrement of temperature, with temperatures </=35 degreesC being critical for the prevention of apoptotic process following transient forebrain ischemia.

Increase of galectin-3 expression in microglia by hyperthermia in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.

Effect of opioids on delayed neuronal death in the gerbil hippocampus.

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented, treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32 degrees C at the end of ischemia produced near-complete protection of CA1 pyramidal neurons. No opioids modulate the protective effect of hypothermia.

Transport of fragmented DNA in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia

Transport of fragmented DNA in apical dendrites of the CA1 pyramidal neurons of gerbil hippocampus is observed in the apoptotic process following transient forebrain ischemia. The time-course of specific DNA fragmentation was examined after the ischemic insult by in situ nick-end-labeling method and fluorescence detection technique by DAPI. Although the role of the fragmented DNA movement is unclear, the transport mechanism of fragmented DNA is still active in the late phase of apoptotic process.

Delta-sleep-inducing peptide (DSIP) stimulates the release of immunoreactive Met-enkephalin from rat lower brainstem slices in vitro

We studied whether delta-sleep-inducing peptide (DSIP) acted on opioid receptor directly or indirectly. DSIP did not have binding activity to any subtype of opioid receptors. DSIP at doses of 1 pM-1 nM significantly stimulated the release of immunoreactive Met-enkephalin (iME) from superfused slices of the rat lower brainstem. The DSIP-induced release of iME was calcium-dependent. These results show that DSIP acts on opioid receptor indirectly by stimulating the release of iME in producing antinociceptive effects.

Prevention of ischemia-induced hippocampal neuronal damage by 2-deoxy-D-glucose in gerbils.

It has been reported that delayed neuronal death (DND) in the hippocampus following transient forebrain ischemia is associated with internucleosomal DNA fragmentation, indicating apoptosis. This suggests that the process of DND is energy dependent. Transient severe forebrain ischemia was induced in Mongolian gerbils by bilateral occlusion of the common carotid arteries. Post-ischemic administration of 2-deoxy-D-glucose (2-DG), a glucose antimetabolite, markedly reduced the occurrence of ischemia-induced DNA fragmentation and DND in the hippocampus. These results suggest that the reduction of energy dependent metabolism after ischemia may be an attractive therapeutic strategy for preserving hippocampal neurons vulnerable to ischemia.