Shutaro Takashima

Inhibition of nitric oxide synthesis induces a significant reduction in local cerebral blood flow in the rat

The effect of intravenous administration of NG-monomethyl-L-arginine (L-NMMA, 30 mg/kg), a specific inhibitor of nitric oxide synthesis, on local cerebral blood flow (lCBF) was examined in the rat using the [14C]iodoantipyrine autoradiographic method. L-NMMA induced a statistically significant reduction in lCBF in the cerebral cortices as well as in various deep structures of the brain. This reduction in lCBF was accompanied by a clear increase in mean arterial blood pressure, suggesting that the cerebral resistance vessels constricted significantly beyond the autoregulatory response following L-NMMA administration. These findings indicate that the basal cerebral circulation may be closely related to nitric oxide production.

Autoradiographic analysis of second-messenger systems in the gerbil brain

Quantitative in-vitro autoradiographic study was performed to localize two prominent second-messenger systems (the adenylate cyclase and phosphoinositide systems) in the normal gerbil brain. [3H] Forskolin and [3H] phorbol 12, 13-dibutyrate (PDBu) were used to identify the regional distribution of adenylate cyclase and protein kinase C, respectively. The localization of the forskolin binding was not uniform, being particularly concentrated in the striatum, the accumbens nucleus, the olfactory tubercle, the substantia nigra, the CA3 region of the hippocampus and the molecular layer of the cerebellum. On the other hand, the PDBu binding was rather uniform, although the superficial layer of the cerebral neocortices, the strata oriens of the CA1 region of the hippocampus and the molecular layer of the cerebellum showed relatively dense binding. Quantitative autoradiography of the second-messenger systems in the brain is expected to provide important information concerning the role of neurotransmitters in the pathophysiology of various conditions.

Autoradiographic analysis on second-messenger systems and local cerebral blood flow in ischemic gerbil brain

Alterations of the second-messenger systems, adenylate cyclase (AC) and protein kinase C (PKC), and local cerebral blood flow (1CBF) were evaluated during experimental cerebral ischemia in gerbils employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. Ischemia was induced by occlusion of the right common carotid artery for 6 h. Animals attaining more than 5 in their ischemic scores were utilized for further experiments. At the end of ischemia, 1CBF was measured by the [14C]iodoantipyrine method. The AC and PKC activities were estimated by the autoradiographic technique developed in our laboratory using [3H]forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu), respectively. The 1CBF fell below 10 ml/100 g/min in most cerebral regions on the ligated side. The greatest reduction in FK binding was noted in the olfactory tubercle, caudate-putamen, and globus pallidus, followed by the hippocampus and cerebral cortices. The FK binding tended to be low at 1CBF < 20 ml/100 g/min in the cerebral cortices. However, the PDBu binding was relatively well preserved in each cerebral structure, and no significant correlation between 1CBF and PDBu binding was noted in the cerebral cortices. The AC system may thus be vulnerable to ischemic insult over extensive brain regions, while the PKC system may be relatively resistant to ischemia.

Effects of lesioning of the substantia innominata on autoregulation of local cerebral blood flow in rats.

Recently, accumulated data have suggested that the nucleus basalis magnocellularis, i.e., the substantia innominata (SI), may represent the primary source of central cholinergic innervation in the rat cortical vasculature. We therefore examined the effects of unilateral lesion of the SI on the autoregulation of local CBF (1CBF) during induced hypotension in rats. Male Wistar rats were divided into three groups. The animals of groups 1 and 2 received an injection of 5 μg of ibotenate into the right SI stereotaxically. At 7 days after the injection, the 1CBF was measured by the [14C]iodoantipyrine technique in the awake state. Group 1 was used as the normotensive group (MABP = 113.1 ± 12.2 mm Hg). Group 2 formed the hypotensive group, and the 1CBF was measured during hypotension (MABP = 80.0 ± 5.5 mm Hg) induced by hemorrhage. Group 3, the sham-operated normotensive group, received vehicle injection into the right SI at 7 days prior to the 1CBF measurement. In group 1, 1CBF was significantly lower in the frontal, parietal, temporal, and striate cortices on the lesioned side compared to that on the contralateral side. In group 2, 1CBF was significantly decreased in the cortices on the lesioned side, but there was no significant difference in magnitude of the 1CBF reduction between groups 1 and 2. Group 3 exhibited no hemispheric asymmetries in 1CBF. These findings suggest that the SI exerts an influence on cortical 1CBF, but does not play a role in the autoregulation of 1CBF during hypotension.

Are blood platelets involved in the pathogenesis of ischemic brain edema in gerbils

Edema formation following severe permanent or temporary cerebral ischemia in gerbils with an artificially reduced platelet count was investigated. Acute focal cerebral ischemia was produced by extracranial carotid ligation, and the local cerebral blood flow was estimated using the hydrogen clearance method. Brain tissue water and sodium and potassium contents were taken as indexes of brain edema. The platelet count was reduced in some gerbils by intravenous injection of neuraminidase. After 60 minutes of ischemia, a marked increase in tissue water and sodium contents accompanied by a decrease in potassium content was observed in untreated gerbils. However, gerbils with a reduced platelet count revealed similar but significantly smaller changes in all the measured parameters. Restoration of blood flow after 60 minutes of ischemia resulted in further accumulation of water and sodium and in depletion of potassium in both groups. These changes were significantly smaller in the gerbils with a reduced platelet count. It is concluded that platelets, activated by cerebral ischemia, may be involved in the development of ischemic brain edema in gerbils.

Alteration of second-messenger ligand binding following 2-Hr hemispheric ischemia in the gerbil brain

The alterations of second-messenger ligand binding and cerebral blood flow (CBF) were evaluated in the gerbil brain after 2-h unilateral common carotid artery occlusion. [3H]Forskolin (FK) and [3H]phorbol-12,13-dibutyrate (PDBu) were used as specific ligands for adenylate cyclase (AC) and protein kinase C (PKC) activity estimation, respectively. CBF was determined at the end of the experiment by the [14C]iodoantipyrine method. A quantitative autoradiographic method permitted simultaneous measurement of the three parameters in the same brain. The levels in the caudate-putamen, globus pallidus, and hippocampus were analyzed. The animals were divided into three groups: Group 1 with severe ischemia (CBF in the lateral nuclei of the thalamus (CBFt) less than 50 ml/100 g/min), Group 2 with mild ischemia (CBFt greater than or equal to 50 ml/100 g/min), and the Sham Group. The PDBu binding revealed a statistically significant increase in the caudate-putamen, lateral nuclei of the thalamus and hippocampus (CA1 and CA3 regions and dentate gyrus) on the ischemic side in Group 1 as compared to that in Group 2 and the Sham Group. In contrast, the FK binding did not show any significant changes in any of the regions. These data and our previous findings for 6-h ischemia suggest that (1) PKC translocation to the cell membrane may occur at the early ischemic phase in particular regions including the caudate-putamen, lateral nuclei of the thalamus and hippocampus, with the translocated PKC gradually diminishing during the subsequent ischemic period; and (2) the suppression of the AC system observed in 6-h ischemia may not appear in the early ischemic phase.

Dual effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils.

The effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils was studied. Administration of naloxone in dose 1 mg/kg to intact gerbils resulted in a marked increase in platelet aggregability accompanied by 27% reduction in cerebral blood flow. Focal cerebral ischemic injury significantly enhanced platelet aggregatory response and treatment with naloxone was without any additional effect on platelet aggregation. Cerebral blood flow in ischemic hemisphere, however, increased following naloxone injection by 46%. In vitro naloxone in millimolar concentrations inhibited platelet aggregation in a dose-dependent way. Apparent decrease in fluorescence of platelet membranes tagged with fluorescence probe due to naloxone suggests conformational changes in platelet membrane as a primary mechanism for the antiaggregatory effect of naloxone in vitro.

Flow threshold for enhanced phorbol ester binding in the ischemic gerbil brain

The correlation between regional phorbol ester binding and cerebral blood flow (CBF) was evaluated in the gerbil brain after 2-hour unilateral common carotid artery occlusion [3H]phorbol 12, 13-dibutyrate (PDBu) was used as a specific ligand for estimating the translocation of protein kinase C (PKC), and CBF was determined by the [14C]iodoantipyrine method. A quantitative autoradiographic method permitted concurrent measurement of these two parameters in the same brain. In the ischemia group of the animals, statistically significant, inverse correlations were noted between the CBF and PDBu binding in the hippocampus (CA1 and CA3 regions and dentate gyrus), the caudate-putamen and lateral nuclei of the thalamus. In these regions, the PDBu binding increased progressively as CBF fell below 35–40 ml/100 g/min. On the other hand, the PDBu binding in the cerebral cortices did not show any significant changes even when CBF was decreased to below 35 ml/100 g/min. The above data suggest that (1) the translocation of PKC to the cell membrane may be regionally specific in response to ischmia and may remain in the regions particularly vulnerable to ischemia such as the hippocampus, caudate-putamen and lateral nuclei of the thalamus in the early ischemic phase; (2) the threshold of CBF below which PKC begins to translocate to the cell membrane in the above regions, may be 35–40 ml/100 g/min in 2-hour ischemia.

Reduction in second-messenger ligand binding sites after brain ischemia--autoradiographic Bmax and Kd determinations using digital image analysis.

Changes in forskolin (FK) and phorbol 12,13-dibutyrate (PDBu) binding were evaluated in relation to local cerebral blood flow (CBF) after 6-h unilateral carotid artery occlusion in the gerbil striatum employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. CBF was measured by the [14C]iodoantipyrine method at the end of the experiment. [3H]FK and [3H]PDBu were utilized as specific ligands to assess the activity of adenylate cyclase (AC) and protein kinase C (PKC), respectively. A saturation study was undertaken to measure the Kd (dissociation constant) and Bmax (maximal binding capacity) of each ligand by digital image processing of sequential autoradiograms employing pixel-by-pixel Scatchard analysis. The Bmax values of FK and PDBu were significantly decreased on the ischemic side, but the reduction in Bmax of FK was greater than that of PDBu. The K4 of each ligand remained unchanged. The FK binding underwent a progressive decline as CBF fell below 30 ml/100 g/min. The PDBu binding showed only a gradual decline in parallel with the CBF reduction. These findings suggest that a reduction in CBF below 30 ml/100 g/min for 6 h may induce a remarkable suppression of the AC system with less significant inhibition of the PKC system in the striatum.

Immobilization stress induces alterations of second-messenger systems in the gerbil brain

The effects of immobilization stress on the cerebral second messenger (adenylate cyclase and phosphoinositide) were investigated autoradiographically in mongolian gerbils. After 10 min (10-min stress group, n = 7), or after 6 h (6-h stress group, n = 7) of fixation on a flat board while supine, in vitro autoradiography was performed using [3H]forskolin (3H-FK) and [3H]phorbol-12,13-dibutyrate (3H-PDBu) as specific ligands to identify the distribution of adenylate cyclase and protein kinase C, respectively. In another group of 7 gerbils (control group), the same autoradiographic procedure was performed immediately after the animals were removed from the cage. In the 10-min stress group, FK binding was significantly decreased in the hypothalamus and amygdala, but significantly increased in the basal ganglia including the caudate-putamen and globus pallidus. FK binding in the 6-h stress group tended to increase throughout the brain, rising significantly in the basal ganglia. PDBu binding in either stress group did not change significantly compared to the control group in any region except the hippocampal CA3 region of the 6-h stress group. Under immobilization stress, the adenylate cyclase system may undergo time-dependent and regionally specific changes, while the phosphoinositide system remains relatively stable.