Taro Kondo

Acute ischemic vulnerability of PKA in the dendritic subfields of the hippocampus CA1

ALTERATIONS of [3H]cyclic AMP (cAMP) binding, an indicator of the binding activity of particulate cyclic AMP-dependent protein kinase (PKA), were examined after 15 and 30 min of ischemia in the gerbil brain. Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. Significant reductions in cAMP binding were noted only in the dendritic subfields of the hippocampus CA1 such as the strata oriens, radiatum and lacunosum-moleculare, on the ischemic side after 15 min of ischemia. After 30 min ischemia cAMP binding was significantly decreased not only in each dendritic subfield of the hippocampus CA1, but also in the layer of pyramidal cell bodies (stratum pyramidale) on the occluded side; other brain regions such as the hippocampus CA3, dentate gyrus and cerebral cortices revealed no significant changes in cAMP binding. These findings suggest that derangement of PKA may begin in the dendritic subfields of the hippocampus CA1 after as little as 15 min of severe ischemia, and proceed centrally to the neuronal cell bodies of the hippocampus CA1.

Calcineurin inhibitor, FK506, prevents reduction in the binding capacity of cyclic AMP-dependent protein kinase in ischemic gerbil brain

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils, FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.

ROLE OF THE RYANODINE RECEPTOR IN ISCHEMIC BRAIN DAMAGE :LOCALIZED REDUCTION OF RYANODINE RECEPTOR BINDING DURING ISCHEMIA IN HIPPOCAMPUS CA1

Abstract1. The ryanodine receptor has recently been shown to play a pivotal role in the regulation of intracellular Ca2+ concentration via Ca2+-induced Ca2+ release (CICR). Effects of ischemia on CICR in the brain tissue, however, remain largely unknown since only a few reports have been published on this subject. In this paper we report on work in this area by our group and review related progress in this field.2. We examined alterations of ryanodine receptor binding and local cerebral blood flow (LCBF) at 15 min, 30 min, and 2 hr after occlusion of the right common carotid artery in the gerbil brain. A quantitative autoradiographic method permitted simultaneous measurement of these parameters in the same brain. The LCBF was significantly reduced in most of the cerebral regions on the occluded side during each time period of ischemia. In contrast, only in the hippocampus CA1 on the occluded side was a significant reduction in ryanodine binding found at 15 min, 30 min and 2 hr after the occlusion.3. These findings suggest that suppression of ryanodine binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of CICR and that this perturbation may be closely associated with the pathophysiological mechanism that leads to the selective ischemic vulnerability of this region.4. Other recent studies have also reported an important role for ryanodine receptors in neuronal injury such as the delayed neuronal death in the hippocampus CA1. These data suggest that derangement of CICR is likely to be involved in acute neuronal necrosis as well as in delayed neuronal death in ischemia.5. Further studies on clarifying the role of CICR in ischemic brain damage are needed in order to develop new therapeutic strategies for stroke patients.

Flow Threshold for Reduction of Cyclic AMP Binding in the Hippocampus CA1 and other Brain Regions during Stroke Development in Gerbils

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g–1 min–1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA3 and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.

Alteration of ryanodine receptor in the hippocampus CA1 after hemispheric cerebral ischemia

Alterations in ryanodine binding and local cerebral blood flow (LCBF) were examined at 30 minutes and 2 hours post-ischemia in the gerbil brain in order to evaluate the influence of cerebral ischemia on the intracellular channels of Ca2+-induced Ca2+ release (CICR). Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. LCBF was measured at the end of the experiment using [14C]iodoantipyrine method, and the ryanodine binding was evaluated in vitro using [3H]ryanodine as a specific ligand for CICR channels. An autoradiographic method developed in our laboratory enabled us to determine both parameters within the same brain. A group of gerbils who underwent a sham procedure served as controls. LCBF was found to be significantly reduced in most of the cerebral regions on the occluded side at both 30 minutes as well as 2 hours post-ischemia. In contrast, a significant reduction in ryanodine binding was noted only in the hippocampus CA1 on the occluded side at 30 minutes and 2 hours after the occlusion. These findings suggest that regionally specific changes of CICR may be the cause of decreased ryanodine binding in the hippocampus CA1, and that these changes may be related to the pathophysiological mechanisms that cause this region to be particularly vulnerable to ischemia.

Chronic transection of post-ganglionic parasympathetic and nasociliary nerves does not affect local cerebral blood flow in the rat.

The role of post-ganglionic parasympathetic nerve fibers from the sphenopalatine ganglion and nasociliary nerve fibers from the trigeminal ganglion in the regulation of basal cerebral blood flow (CBF) was examined using rats, which had been divided into three groups; a sham group, a denervation group and a denervation+NG-monomethyl-L-arginine (L-NMMA) group. In the denervation and denervation+L-NMMA groups, unilateral chronic transection of the above nerve fibers had been performed at the ethmoidal foramen (EF) for 2 weeks. In the sham group, the above nerve fibers were only exposed at EF and not severed 2 weeks before the CBF measurement. Local CBF was measured by the [14C]iodoantipyrine autoradiographic method after intravenous administration of saline in the sham and denervation groups or L-NMMA (30 mg/kg) in the denervation+L-NMMA group. No significant difference in CBF was noted on each side in any of the regions between the sham and denervation groups. L-NMMA induced a significant reduction in local CBF on either side in each brain region. Neither the animals which were administered saline nor those with L-NMMA showed any side-to-side differences in local CBF in any of the brain regions examined. These findings suggest that the perivascular nerve fibers running through the EF, which are known to contain substantial nitric oxide synthase (NOS), may not play a pivotal role in the regulation of basal CBF. The reduction in CBF induced by the acute administration of L-NMMA was not affected by the chronic denervation of the above NOS-containing perivascular nerves.

Binding capacity of FK506 binding protein after 2-hour hemispheric ischemia in gerbil brain

The binding capacity of FK506 binding protein (FKBP) was examined after 2-h hemispheric ischemia in the gerbil brain in order to clarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal gerbil brain using 1 nM [3H]dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner, although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites of FK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an ischemia group (n = 12) and a sham group (n = 6). The right common carotid artery was ligated to induce hemispheric ischemia for 2 h in the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the [14C]iodoantipyrine method. The ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 ml/100 g/min were utilized as the ischemia group (n=6) for further data analysis. No significant differences in FK506 binding between the ischemia and sham groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-h ischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase of cerebral ischemia.

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.

Role of Nitric Oxide in Autoregulation of Cerebral Blood Flow in the Rat

We examined the role of nitric oxide (NO) in autoregulation of cerebral blood flow (CBF) in the rat. Autoregulation of the CBF is defined as the physiological tendency of the brain to maintain a constant CBF despite changes in arterial blood pressure (ABP). Male Sprague-Dawley rats were divided into six groups: a saline group (n = 9), a saline + hypotension group (n = 8), an N G-monomethyl-l-arginine (L-NMMA) group (n = 7), an L-NMMA + hypotension group (n = 9), a denervation group (n = 6), and a denervation + hypotension group (n = 15). We employed the [14C]iodoantipyrine method to measure CBF. In each hypotension group, the ABP was lowered by withdrawing blood. In each L-NMMA group, 30 mg/kg of L-NMMA, a potent NO synthase (NOS) inhibitor, was injected intravenously before making the CBF measurement. In each denervation group, unilateral chronic transection of the perivascular NOS-containing nerve fibers was performed at 2 weeks before the CBF measurement. We found a significant impairment of autoregulation in the L-NMMA and L-NMMA + hypotension groups, whereas the saline and saline + hypotension groups as well as the denervation and denervation + hypotension groups did not show any definite disturbance of autoregulation. We infer that NO, probably derived from the vascular endothelium, may play an important role in autoregulation of CBF.