Multi-focal delayed neuronal damage after transient regional ischemia--mechanism of vascular dementia

We describe multi-focal delayed neuronal death of rat brain after transient regional ischemia induced by embolization of the right middle cerebral artery (MCA). After sixty minutes 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 examination. Sixty minutes after MCA occlusion, 45Ca accumulation extended to the lateral segment of the caudate putamen and the cerebral cortex supplied by the occluded MCA. Moreover, three days after ischemic insult, 45Ca had accumulated in the ipsilateral thalamus and substantia nigra. Histological examination revealed that the neurons in both area suffered damage and were selectively reduced in number. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. We suggest that the postischemic delayed neuronal death in exo-focal remote areas may be caused by a transsynaptic process associated with the infarcted areas and that these delayed multi-focal brain damage may exacerbate clinical symptoms in the chronic stage of stroke.     

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.     

Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after local cerebral ischemia in rat

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2–48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24–48 h after ischemia, neurons in the ischemia lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.     

Regional cerebral blood flow after occlusion of the middle cerebral artery

Occlusions of the middle cerebral artery (MCA) are mostly of embolic origin (appr. 80%) and give rise to about one third of all ischemic strokes, most of these being major strokes. MCA occlusions lasting for less than 1/2 h are tolerated without occurrence of permanent tissue damage. Occlusions lasting between 1/2 h to 4-8 h lead to permanent tissue damage and neurological deficits that are proportional to the duration of occlusion. Maximal tissue damage is obtained after 4-8 h occlusion. A cerebral blood flow of 8-23 ml/100 gr/min is sufficient for cellular viability but insufficient for normal tissue function ("ischemic penumbra"). Cellular function is completely abolished in the interval 8-16 ml/100 gr/min and flow at that level is tolerated only for 1-3 h before neuronal death ensues. In the interval 18-23 ml/100 gr/min there is some functional activity although it is reduced. Experimental and clinical evidence suggests that flow in this interval may be tolerated for several days, months or even longer ("chronic ischemic penumbra"). After MCA occlusion the blood flow falls below 8 ml/100 gr/min in most cases and permanent MCA occlusion always leads to relatively large areas of frank infarction. The ischemic infarcts may be surrounded by collaterally perfused areas where the blood flow is pressure-dependent (impaired autoregulation) and quite commonly insufficient for normal neuronal function (below 23 ml/100 gr/min). Such collaterally perfused areas may include a "chronic ischemic penumbra". Emboli causing MCA occlusions commonly disintegrate and/or migrate more peripherally within the first few weeks post stroke. This leads to reperfusion and changes of ischemic infarcts into hyperemic infarcts where flow is severely increased. The vascular reactivity is completely abolished in hyperemic infarcts and the hyperemic state lasts for about two weeks. Probably, anemic infarcts are equivalent to ischemic infarcts while the hemorrhagic variety is equivalent to hyperemic infarcts. The "partial infarct" with selective neuronal necrosis occurs in experimental animals after MCA occlusions of less than four h but not after permanent MCA occlusion. The significance of partial infarction in human stroke is not clarified. The extent of irreversible tissue damage can be reduced only if therapy sets in within 4-8 h after the occlusion. If a "chronic penumbra" exists the extension of reversible tissue damage can be reduced if therapy aimed at increasing the blood flow in the penumbra sets in within weeks or even months after the stroke.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exo-focal neuronal death in the rat brain

We describe delayed neuronal damage in ipsilateral remote areas outside the ischemic area of rat brain after transient focal ischemia. The distribution of the neuronal damage was determined by using the 45Ca autoradiographic technique and the histological method, and we investigated the mechanism involved by measuring local cerebral glucose metabolism. Wistar rats were used throughout the experiments. Under 2% halothane anesthesia with a mixture of 70% N2O and 30% O2, the right middle cerebral artery (MCA) was embolized by insertion from the internal carotid artery of a nylon surgical thread with a cylindrical coating of silicone on the distal portion. Animals were divided into 4 groups based on duration of ischemia. After 15, 30, 60 and 90 min of MCA occlusion, recirculation was achieved by removal of the embolus. Immediately after recirculation and then after 24 hr, 3 days, 1 week and 2 weeks of recirculation, 300 microCi 45CaCl2 in aqueous solution (0.3 ml) was administered intravenously; 6 hr later, animals were decapitated to obtain autoradiograms. Histological examination was carried out according to the same protocol. In the 15-min MCA occlusion group, neither 45Ca accumulation nor histological change was observed. In the 30-min MCA occlusion group, 45Ca accumulation extended from the lateral margin to the lateral segment of the caudate-putamen and the cerebral cortex supplied by the occluded MCA depending on the duration of recirculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil elastase inhibition reduces cerebral ischemic damage in the middle cerebral artery occlusion

It has been reported that activated neutrophils are involved in the development of cerebral damage induced by ischemia. Activated neutrophils release a lot of mediators including toxic oxygen metabolites, elastase and cytokines which damage brain tissue. Therefore, we investigated roles of neutrophil elastase in the development of cerebral damage using an elastase inhibitor, ONO-5046. The rat middle cerebral artery (MCA) was occluded by a thrombus induced by photochemical reaction between green light and the photosensitizer dye, Rose Bengal. Photochemical reaction causes endothelial injury followed by formation of a platelet and fibrin-rich thrombus at the site of the irradiation. Photochemical reaction is routinely used in our laboratory to produce arterial occlusion in experimental animals. Twenty-four hours after the MCA occlusion, the size of cerebral damage was measured by histochemical technique. Water content in the brain was measured and neuronal deficits were examined 24 h after the MCA occlusion. ONO-5046 was administered at various doses as continuous infusion for 24 h, starting just after the MCA occlusion or from 3 h after. ONO-5046 at doses of 10 and 30 mg/kg/h significantly (p<0.05 and p<0.01, respectively) reduced the size of cerebral damage and water content (p<0.05, p<0.01, respectively) in different eight rats. Further, ONO-5046 at a dose of 30 mg/kg/h significantly (p=0.01) improved neuronal deficits. ONO-5046 which was administered starting from 3 h after the MCA occlusion, also reduced the size of cerebral damage. Neutropenia by anti-neutrophil antibody injection significantly (p<0. 01) reduced the size of cerebral damage. Elastase released from activated neutrophils may play a key role in the development of cerebral damage.     

Morphological consequences of early reperfusion following thrombotic or mechanical occlusion of the rat middle cerebral artery

Summary The early morphological consequences of recirculation following middle cerebral artery (MCA) occlusion were studied in two rat models. The proximal MCA was occluded for 1 h by either a surgical clip or platelet thrombus; subsequently, 1 h of recirculation was facilitated. Following clip occlusion and recirculation, mild astrocytic swelling, especially around blood vessels, was detected in reperfused cortical and striatal areas. Neuronal changes included slight chromatin clumping and dilation of the rough endoplasmic reticulum. In contrast, severe structural abnormalities were detected following recanalization of the thrombosed MCA segment. Marked astrocytic swelling of cell bodies and perivascular processes with neuropil vacuolation were commonly seen. A heterogeneous pattern of neuronal alterations, including a high frequency of dense shrunken neurons surrounded by swollen astrocytic processes was documented in cortical and striatal regions. Severe neuronal changes were documented in brain regions exhibiting a wellperfused microcirculation. Vascular endothelial cells contained large numbers of pinocytotic vesicles associated with luminal and abluminal surfaces. Pronounced and rapid morphological changes evolve with reperfusion when thrombotic and ischemic events occur simultaneously. The basis for these rapid parenchymal changes following vascular thrombosis may involve acute alterations in cerebral microvascular permeability which exacerbate ischemic consequences.Supported by USPHS grants NS-05820 and NS-23244, National Parkinson Foundation Grant YR 660068, and by the American Heart Association Grant-in-Aid 87-1012, with funds contributed by the Florida Affiliate. W. Dalton Dietrich is an Established Investigator of the American Heart Association.     

Nimodipine and the haemodynamic and histopathological consequences of middle cerebral artery occlusion in the rat

The effect of the administration of nimodipine (1 microgram kg-1 min-1), initiated 5 min after occlusion of a middle cerebral artery (MCA), upon cerebral haemodynamics ([14C]iodoantipyrine autoradiography) and neuropathological outcome (volume of histologically ischaemic tissue) was investigated in anaesthetized rats. Measurements were made of the level of local CBF (LCBF) in a total of 37 neuroanatomically defined areas, either ipsilateral or contralateral to the occluded vessel, and the autoradiograms were examined using a new approach to quantitative densitometry that employed a frequency distribution analysis of the CBF in sections of the brain at different coronal planes. Both methods of analysis showed that nimodipine, administered after the ischemic event, did not modify the pattern of CBF distribution after MCA occlusion. The extent of ischaemic brain damage was determined by histological examination. There was no evidence that the extent of ischaemic damage, either in sections at eight different coronal planes or in overall volume, was significantly different in animals that received nimodipine from that observed in animals that received only the vehicle used to dissolve the drug. The lack of cerebral circulatory and neuropathological alterations when nimodipine administration is initiated after occlusion of the MCA is contrasted with the higher levels of LCBF and the reductions in the volume of ischaemic tissue that were found when nimodipine was administered before occlusion of the artery.     

An immunohistochemical study of copper/zinc superoxide dismutase and manganese superoxide dismutase following focal cerebral ischemia in the rat

We investigated immunohistochemically the localization and changes of copper/zinc superoxide dismutase (CuZn-SOD) and manganese superoxide dismutase (Mn-SOD) in the rat brain following 1 h of middle cerebral artery (MCA) occlusion. In normal brain, immunoreactivity to both SODs was observed in medium-sized neurons in the striatum and in many neurons in the neocortex. Mn-SOD was predominantly stained in cortical interneurons. The immunostaining of both SODs rapidly decreased or disappeared in neurons in the lateral segment of the striatum (ischemic center) 4 h after MCA occlusion, when the neurons were degenerating. Most neurons in the neocortex (ischemic penumbra) decreased their CuZn-SOD immunoreactivity but not Mn-SOD immunoreactivity 4 h after ischemia, when only a few neurons showed histopathological changes. CuZn-SOD immunoreactivity in almost all cortical neurons disappeared 1 day after ischemia, but Mn-SOD immunoreactivity was still preserved in interneurons, when cortical neurons showed typical pathological changes. Some cortical neurons in the boundary zone between normal and infarcted areas showed intense immunostaining to both SODs and glial SOD immunoreactivity appeared after 3 and 7 days. These results suggest that early loss of the scavenging system of free radicals may lead to neuronal damage after ischemic insult, and that induced SODs in the boundary zone between the normal and infarcted areas may act as a defense mechanism against damage.     

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.     

Inflammatory response and neuronal necrosis in rats with cerebral ischemia

In the middle cerebral artery occlusion model of ischemic injury, inflammation primarily occurs in the infarct and peripheral zones. In the ischemic zone, neurons undergo necrosis and apoptosis, and a large number of reactive microglia are present. In the present study, we investigated the pathological changes in a rat model of middle cerebral artery occlusion. Neuronal necrosis appeared 12 hours after middle cerebral artery occlusion, and the peak of neuronal apoptosis ap- peared 4 to 6 days after middle cerebral artery occlusion. Inflammatory cytokines and microglia play a role in damage and repair after middle cerebral artery occlusion. Serum intercellular cell adhesion molecule-1 levels were positively correlated with the permeability of the blood-brain barrier. These findings indicate that intercellular cell adhesion molecule-1 may be involved in blood-brain barrier injury, microglial activation, and neuronal apoptosis. Inhibiting blood-brain barrier leakage may alleviate neuronal injury following ischemia,     

Blockade of central histaminergic H2 receptors facilitates catecholaminergic metabolism and aggravates ischemic brain damage in the rat telencephalon

Blockade of central H(2) receptors aggravates ischemic neuronal damage. Since changes in the activity of the monoaminergic system are contributing factors in the development of ischemic neuronal damage, the authors evaluated the effects of ranitidine on the monoaminergic system and ischemic neuronal damage in the middle cerebral artery (MCA) occlusion model of rats. Wistar rats pretreated with saline or ranitidine (3 and 30 nmol, i.c.v.) were subjected to reversible occlusion of MCA for 2 h. The total infarct volume was determined 24 h after reperfusion. The relationship between dopaminergic activity and the histologic outcome was estimated by lesioning the substantia nigra 2 days before MCA occlusion. In a second experiment, the animals were subjected to 15 min of MCA occlusion, and the effects of ranitidine on the histologic outcome was evaluated 7 days after ischemia. In a third experiment, the tissue concentrations of monoamines and their metabolites were determined in the cerebral cortex and striatum 2 h after reperfusion following MCA occlusion for 2 h. The turnover of norepinephrine and dopamine was compared between animals treated with saline and those treated with ranitidine by estimating the alpha-methyl-p-tyrosine-induced depletion of norepinephrine and dopamine, respectively. The turnover of 5-hydroxytryptamine was evaluated by the probenecid-induced accumulation of 5-hydroxyindoleacetic acid. Treatments with ranitidine markedly increased the infarct volume 24 h after reperfusion. Ranitidine also aggravated delayed neuronal death 7 days after ischemia. The aggravation was abolished by the lesion of the substantia nigra before MCA occlusion. The MCA occlusion increased the turnover of cortical norepinephrine and striatal dopamine. The turnover was further facilitated by ranitidine. Although ranitidine suppressed the 5-hydroxytryptamine turnover in the cerebral cortex, the extent of this effect was similar in both the ischemic and non-ischemic sides. These results suggest that facilitation of the catecholaminergic systems is involved in the aggravation of ischemic neuronal damage by H(2) blockade.     

Photothrombotic occlusion of rat middle cerebral artery: histopathological and hemodynamic sequelae of acute recanalization

The histopathological and hemodynamic consequences of photochemically induced middle cerebral artery (MCA) thrombosis and recanalization were studied in the rat. Recanalization of the thrombosed MCA segment was achieved by the topical application of nimodipine at 1 h following photochemically induced occlusion. Pathological consequences of permanent and temporary occlusion were compared by morphometric procedures 7 days following thrombus formation. Rats with permanent thrombosis exhibited consistent infarction of both striatum and cortex. MCA recanalization at 1 h was associated with a significant reduction in total infarct volume. In recanalized rats, small cortical infarcts, confined to the peripheral MCA territory, were observed in only three of six rats. In contrast, a mixed pattern of infarction and ischemic cell damage was documented throughout the striatum in all rats. Local CBF (1CBF), measured autoradiographically, was significantly reduced in the MCA territory following 1 h of MCA occlusion, especially within the striatum. At 1 h after recanalization, 1CBF recovered within the previously ischemic brain regions to greater than 50% of control. Perfusion deficits were detected by carbon black infusion within focal areas of the striatum following reperfusion. Thus, cortical neurons appear to tolerate 1 h of MCA occlusion in this model. In contrast, reperfusion following 1 h of photochemically induced MCA occlusion gives rise to selective injury to the striatum.     

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.     

Exo-focal postischemic neuronal damage in the rat brain: alteration of [H]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 substantia 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.     

Effect of the kappa-1 opioid agonist CI-977 on ischemic brain damage and cerebral blood flow after middle cerebral artery occlusion in the rat

The effects of the kappa-1 opioid agonist CI-977 upon the volume of ischemic brain damage (defined using quantitative neuropathology) and local cerebral blood flow (CBF) (defined using quantitative [¹⁴C]iodoantipyrine autoradiography) have been examined at 4 h and 30 min, respectively, after permanent middle cerebral artery (MCA) occlusion in halothane-anesthetised rats. Treatment with CI-977 (0.3 mg/kg, s.c.) 30 min before and 30 min after occlusion of the MCA reduced the volume of infarction in the cerebral hemisphere (reduced by 27% when compared to vehicle;P<0.05) and cerebral cortex (reduced by 32%;P<0.05), despite a marked and sustained hypotension, with only minimal effect on damage in the caudate nucleus. In the hemisphere contralateral to the occluded MCA, treatment with CI-977 (0.3 mg/kg, s.c.) 30 min prior to the induction of ischemia failed to demonstrate any significant effect on either the level of local CBF in any of the 25 regions examined or on the volume of low CBF determined by frequency distribution analysis. In the hemisphere ipsilateral to MCA occlusion, CI-977 failed to produce statistically significant alterations in either the level of local CBF in 23 of the 25 regions or on the volume of low CBF, but areas of hyperemia were observed in both the medial caudate nucleus and lateral thalamus (local CBF increased by 65% and 86%, respectively, when compared to vehicle). The results of the present study indicate that the kappa-1 opioid agonist CI-977 is neuroprotective in a rat model of focal cerebral ischemia where key physiological variables have been assessed throughout the entire post-ischemic period, and fail to demonstrate that the neuroprotective effects of CI-977 in this model are due to improved blood flow to ischemic tissue.     

Neuronal injury in the infarct border: a neuropathological study in the rat

Summary Focal ischemia was induced in rats by occlusion of the middle cerebral artery (MCA). Infarction developed primarily in the basal ganglia. The development, density and distribution of neuronal injury in the cortex adjacent to the infarct were studied from 4 h to 1 year after occlusion of the artery. The brains were perfusion fixed, sub-serially sectioned, and stained with H + E, acid fuchsin/cresyl violet, and ad modum Klüver-Barrera. The number of injured neurons was assessed by direct visual counting. Four hours after the artery occlusion, the infarct was clearly outlined in the corpus striatum, whereas the cortical border became sharp 1 to 2 days after ligation of MCA. After 1 day triangular injured neurons with eosinophilic cytoplasm and pyknotic nuclei were seen outside the infarct. The number of injured neurons at day 1, 2, 3, 4, and 5 was the same, i. e. no evidence for delayed neuronal death was found. Neuron counts at day 1, 4, 10, 17, 27, and 365 were reduced according to the number of acutely injured neurons. Most injured neurons were observed less than 3 mm from the margin of the infarct and the greatest number was found in the cortical layers 2 and 3. The border zone in the medial part of the striatum showed selective neuronal necrosis only in a zone of 200 m. The fact that the number of injured neurons was constant from day 1 to 5 after artery occlusion indicates that the damage is acute and that a delayed loss of neurons is of minor significance.     

Therapeutic time window of tacrolimus (FK506) in a nonhuman primate stroke model: comparison with tissue plasminogen activator

Tacrolimus (FK506), an immunosuppressive drug, has been shown to exert a potent neuroprotective activity when administered immediately after occlusion of the middle cerebral artery (MCA) in a nonhuman primate model of stroke. Here, we assessed the neuroprotective efficacy of tacrolimus with delayed treatment using the same model and compared with that of recombinant tissue plasminogen activator (rt-PA). Ischemic insult was induced by photochemically induced thrombotic occlusion of MCA in cynomolgus monkeys, and tacrolimus (0.2 mg/kg) and/or rt-PA (1.0 mg/kg) was intravenously administered 2 h after MCA occlusion. In another experiment, tacrolimus (0.1 mg/kg) was administered 4 h after MCA occlusion. Neurological deficits were monitored for 28 days after the ischemic insult and cerebral infarct volumes were measured with brain slices. With drug administration 2 h after the ischemic insult, tacrolimus significantly reduced neurological deficits and infarct volumes in the cerebral cortex without affecting the recanalization pattern in the MCA, however, rt-PA did not significantly improve neurological deficits or infarct volumes, even though it increased the recanalization rate of the occluded MCA. Combined treatment with tacrolimus and rt-PA exerted additional protection. Administration of tacrolimus 4 h after the ischemic insult still showed significant amelioration of neurological deficits. These results suggested that tacrolimus had a wider therapeutic time window than rt-PA in the nonhuman primate stroke model.     

Cilostazol reduces ischemic brain damage partly by inducing metallothionein-1 and -2

The neuroprotective effect of cilostazol, an antiplatelet drug, was examined after 24 h permanent middle cerebral artery (MCA) occlusion in mice, and explored the possible underlying mechanism by examining metallothionein (MT)-1 and -2 induction in vivo. Cilostazol (30 mg/kg) was intraperitoneally administered at 12 h before, 1 h before, and just after MCA occlusion. Mice were euthanized at 24 h after the occlusion, and the neuronal damage was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cilostazol significantly reduced the infarct area and volume, especially in the cortex. Real-time RT-PCR revealed increased mRNA expressions for MT-1 and -2 in the cortex of normal brains at 6 h after cilostazol treatment without MCA occlusion. MT-1 and -2 immunoreactivity was also increased in the cortex of such mice, and this immunoreactivity was observed in the ischemic hemisphere at 24 h after MCA occlusion (without cilostazol treatment). The strongest MT-1 and -2 immunoreactivity was detected in MCA-occlused mice treated with cilostazol [in the peri-infarct zone of the cortex (penumbral zone)]. These findings indicate that cilostazol has neuroprotective effects in vivo against permanent focal cerebral ischemia, especially in the penumbral zone in the cortex, and that MT-1 and -2 may be partly responsible for these neuroprotective effects.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. 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 hours 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]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.