Haruo Nagasawa

Exo-focal postischemic neuronal death in the rat brain

We describe delayed neuronal damage in ipsilateral areas remote from the ischemic area of rat brain after transient focal ischemia induced by embolization of the right middle cerebral artery (MCA). After 15, 30, 60 and 90 min of MCA occlusion, recirculation was achieved by removal of the embolus. Chronological changes in the distribution of the neuronal damage were determined by using the 45Ca autoradiographic technique and the histological method, and the mechanism involved was investigated by measuring local cerebral glucose metabolism. Depending on the duration of ischemia, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. Moreover, 3 days after ischemic insult, 45Ca had accumulated in the ipsilateral substantia nigra and ventral posterior nucleus of the thalamus. Histological examination revealed that the neurons in both areas suffered damage and were selectively reduced in number. Cerebral glucose utilization decreased in the thalamus, but increased approximately 30% (P less than 0.01) in the substantia nigra compared with the value in the corresponding contralateral area. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. Based on the present study, we suggest that the mechanisms of delayed neuronal death in these two remote areas may not be identical, but that this phenomenon may be caused by a transsynaptic process associated with the ischemic focus.

Behavioral studies on rats with transient cerebral ischemia induced by occlusion of the middle cerebral artery

The behavioral effects of transient cerebral ischemia in adult Wistar rats were studied. In Experiment 1, rats were subjected to 90-min occlusion of the unilateral, left or right, middle cerebral artery (MCA) followed by recirculation. The locomotor activity had not changed 3 and 30 days after the occlusion, except that the number of rearing was significantly decreased by left MCA occlusion. Rats were examined in a radial maze system for learning and memory ability during 4 weeks from the 3rd day after ischemia (the 3rd day was counted as day 1 of the experiment). Maze performance was slightly disturbed due to focal brain damage by MCA occlusion, but the disturbance was statistically significant only on days 6, 11, and 15 in the right occlusion. In Experiment 2, rats were trained to master a radial maze task completely for 4 weeks, and then subjected to transient unilateral (right) ischemia as described above. These rats showed an increase in incorrect entry in the radial maze task from day 4 to day 14. However, on day 21, the number of incorrect entry decreased to the control level of the sham-operated group. The numbers of correct choice were inversely related with those of incorrect entry, though slightly blunted. Coincidentally, the time required to solve the maze task was also prolonged from day 4 to day 14, but returned to the control time on day 21. These results suggest that unilateral ischemia transiently suppresses both acquiring radial maze performance and maintenance of learned performance and that it is a good model for studying human focal cerebral ischemia.

PET study of cerebral glucose metabolism and fluorodopa uptake in patients with corticobasal degeneration

We measured cerebral glucose utilization and fluorodopa metabolism in the brain of patients with corticobasal degeneration using position emission tomography. The clinical pictures are distinctive, comprising features referable to both cerebral cortical and basal ganglionic dysfunctions. Brain images of glucose metabolism can demonstrate specific abnormalities with a marked asymmetry in the parietal cortex (the primary motor and sensory cortex and the lateral parietal cortex), the thalamus, the caudate nucleus and the putamen of the dominantly affected hemisphere related to clinical symptoms in six patients. [18F]dopa uptake also reduced in an asymmetric pattern, both the caudate nucleus and the putamen in four patients. This unique combination study measuring both cerebral glucose utilization and fluorodopa metabolism in the nigrostriatal system can provide efficient information about the dysfunctions which are correlated with individual clinical symptoms.

Alteration of adenosine A1 receptor binding in the post-ischaemic rat brain.

Chronological changes of adenosine A, receptor binding of the rat brain were determined by in vitro [3H]cyclohexyladenosine (CHA) autoradiography after 90 min of middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. One day after the ischaemia, [5H]CHA binding sites decreased significantly in the cerebral cortex (p < 0.05) and lateral segment of the caudate putamen (p < 0.01), both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischaemic foci. On the contrary, there, was no alteration on day 1, but 3 days after ischaemic insult, a significant decrease of [3H]CHA binding sites was first detected in the ipsilateral thalamus and the substantia nigra, both areas which had not been directly affected by the original ischaemic insult. This post-ischaemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with 45Ca accumulation, which was studied in our previous study. Based on the present study, alteration of adenosine A, receptor binding activities is involved not only in the ischaemic foci, but also in the remote areas associated neuroanatomically with the ischaemic foci. We suggest that multi-focal post-ischaemic alterations of adenosine A1 binding activities may exacerbate clinical symptoms of patients at a chronic stage of stroke.

Metabolic disturbances in exo-focal brain areas after cortical stroke studied by positron emission tomography

We have reported that exo-focal delayed neuronal damage was observed in the ipsilateral thalamus and the substantia nigra of the rat brain after occlusion of the middle cerebral artery (MCA). To determine if that phenomenon also occurs in humans, we measured cerebral metabolic rates for glucose (CMRGlc) in the remote brain areas at a chronic stage after cortical infarction using 2-[18F]fluoro-2-deoxy-D-glucose and position emission tomography (PET). The subjects studied were 11 patients who were affected by unilateral cerebral infarction in the cortex supplied by MCA. There were significant decreases of CMRGlc as compared with control values (p < 0.01), not only in the cerebral cortex directly damaged by the ischemic insult, but also in the ipsilateral thalamus and in the contralateral cerebellum, areas in which no lesions had been detected by MRI or CT scan. The present study indicates that different mechanisms may be responsible for multi-focal metabolic disturbances in the remote areas after stroke. The reduction of CMRGlc in the contralateral cerebellum may be explained by the crossed cerebellar diaschisis theory and in the ipsilateral thalamus as being due to retrograde degeneration associated with the infarcted cortex. We suggest that these multi-focal brain dysfunctions caused by neuronal network disturbances may exacerbate clinical symptoms at a chronic stage of stroke.

6-[18F]Fluorodopa metabolism in patients with hemiparkinsonism studied by positron emission tomography

A group of 10 healthy control subjects and 10 patients with hemiparkinsonism (HD) were studied by positron emission tomography (PET) using 6-[18F]fluorodopa (FDOPA). FDOPA metabolism in the caudate nucleus and the putamen was separately estimated by measuring target-to-background ratios (TBRs) using composite images added between 30 and 60 min after FDOPA injection and by TBR-versus-time slopes during PET study. TBRs in the caudate nucleus and the putamen were 1.81 +/- 0.23 (mean +/- SD) and 1.92 +/- 0.28 in the 10 controls, respectively. In HD patients, on the dominantly affected hemisphere related to main clinical symptoms, TBRs were significantly decreased in the caudate nucleus (P < 0.01) and the putamen (P < 0.05) compared with those in the corresponding areas on the contralateral hemisphere, though those TBRs on both hemispheres were significantly decreased compared with the TBRs of normal subjects (P < 0.01). TBRs and TBR slopes in both the caudate nucleus and the putamen were correlated with disease severity according to Hoehn and Yahr. On the dominantly affected hemisphere, TBR and TBR slopes in the putamen were well correlated with individual clinical measures for bradykinesia and rigidity, and those in the caudate nucleus were also correlated with the severity of tremor. Our data suggest that in HD patients, PET study using FDOPA may provide unique and efficient information on the dysfunction of the dominantly affected caudate nucleus and the putamen which are correlated with diseased severity and individual clinical symptoms.

Alteration of muscarinic acetylcholine binding sites in the postischemic brain areas of the rat using in vitro autoradiography

We studied the postischemic alteration of muscarinic acetylcholine binding sites in the rat brain using in vitro [3H]quinuclidinyl benzilate (QNB) autoradiography. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 days of recirculation, [3H]QNB binding sites were found to be significantly decreased in the cerebral cortex (P < 0.01) and lateral segment of the caudate putamen (P < 0.05), both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. Moreover, 3 days after the ischemia, significant decreases of [3H]QNB binding sites were observed in the ipsilateral thalamus and the amygdala, and also in the substantia nigra 1 week after the ischemia, areas which had not been directly affected by the original ischemic insult. This postischemic phenomenon observed in the thalamus and the substantia nigra developed concurrently with 45Ca accumulation, which was detected there in our previous study. These results indicate that alteration of muscarinic acetylcholine binding sites may be involved not only in the ischemic foci, but also in the exo-focal remote areas, in which delayed neuronal degeneration due to neuronal network disturbances after the ischemia was observed. We suggest that multifocal postischemic alterations of muscarinic acetylcholine binding sites may exacerbate the clinical symptoms of patients during the chronic stage of stroke.

Exo-focal postischemic neuronal damage in the rat brain: alteration of [3H]forskolin binding using in vitro autoradiography

We studied the alteration of intracellular signal transduction using quantitative autoradiography of the second messenger system in order to clarify the mechanisms of delayed neuronal damage in the remote areas of rat brain after transient focal ischemia. Chronological changes of [3H]forskolin binding sites were measured to demonstrate the striatal-nigral pathway after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by 3 h, 6 h, 1 day, 3 days, 1 week, 2 weeks and 4 weeks of recirculation. [3H]Forskolin binding sites were found to be markedly decreased in the lateral segment of the caudate putamen supplied by the occluded MCA after 90 min of ischemia with no recirculation. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, marked reduction of [3H]forskolin binding sites was observed in the ipsilateral substantial nigra which lay outside the ischemic areas. This postischemic delayed phenomenon observed in the substantia nigra developed concurrently with 45Ca accumulation, which was detected there in our previous study. The delayed reduction of [3H]forskolin binding sites in the substantia nigra observed in the present study indicates that striatonigral terminal degeneration at presynaptic sites is caused by precedent ischemic damage of the ipsilateral caudate putamen and that exo-focal postischemic neuronal death is caused by a transsynaptic process associated with the ischemic foci.

Brain 6-[18F]fluorodopa metabolism in early and late onset of Parkinson's disease studied by positron emission tomography.

We measured 6-[18F]fluorodopa (FDOPA) uptake in the caudate nucleus and the putamen of 20 patients with early and late onset of Parkinsons disease (EOPD and LOPD) and 20 normal control subjects using positron emission tomography. The mean influx rate constant values (Ki) were significantly reduced in the caudate nucleus and the putamen of the patients with EOPD and LOPD compared with age-matched control groups (p < 0.01), respectively. There were significant negative correlations between Ki values in the caudate nucleus (r = -0.67, p = 0.0024) and the putamen (r = -0.67, p = 0.0014), and duration of disease in the LOPD group compared with the EOPD group. Similar negative relationships between Ki values and clinical stages by Hoehn and Yahr and degrees of main clinical symptoms (bradykinesia, tremor and rigidity) were more markedly seen in the LOPD group than in the EOPD group. The present results suggest that the function of presynaptic dopaminergic terminals correlates well with clinical disease severity and degrees of main symptoms in the LOPD group, but not in the EOPD group. We speculate that compensatory up-regulatory function in the postsynaptic dopaminergic receptors may modify disease severity and the degrees of main clinical symptoms of EOPD.

Autoradiographic analysis of second-messenger and neurotransmitter receptor systems in the exo-focal remote areas of postischemic rat brain

We studied the chronological changes of protein kinase C (PKC) and muscarinic acetylcholine receptor binding activities of the rat brain which were determined by using [3H]phorbol 12,13-dibutyrate (PDBu) and [3H]quinuclidinyl benzilate (QNB) autoradiographic methods, respectively, after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion, followed by different periods of recirculation. After the ischemic insult followed by 3 h of recirculation, [3H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [3H]QNB binding sites was first detected in those ischemic foci. Moreover, 3 days after ischemic insult, both [3H]PDBu and [3H]QNB binding sites were concurrently reduced in the ipsilateral thalamus and 1 week after the ischemia, in the substantia nigra, in which both areas had not been directly affected by the original ischemic insult. These alterations of PKC in the postischemic brain areas developed concurrently with 45Ca accumulation, which was detected in our previous study. These results suggest that postischemic alterations of second-messenger (PKC) and neurotransmitter receptor systems were involved not only in the ischemic foci due to ischemia-induced energy failure, but also in the exo-focal remote areas prior to the histologic changes where neuronal damage might be caused by transsynaptic delayed degeneration.