Alterations of potassium currents in ischemia-vulnerable and ischemia-resistant neurons in the hippocampus after ischemia

CA1 pyramidal neurons in the hippocampus die 2-3 days following transient forebrain ischemia, whereas CA3 pyramidal neurons and granule cells in the dentate gyrus remain viable. Excitotoxicity is the major cause of ischemic cell death, and potassium currents play important roles in regulating the neuronal excitability. The present study compared the changes of potassium currents in acutely dissociated hippocampal neurons at different intervals after ischemia. In CA1 neurons, the amplitude of rapid inactivating potassium currents (I(A)) was significantly increased at 14 h and returned to control levels at 38 h after ischemia; the rising slope and decay time constant of I(A) were accordingly increased after ischemia. The activation curve of I(A) in CA1 neurons shifted to the depolarizing direction at 38 h after ischemia. In granule cells, the amplitude and rising slope of I(A) were significantly increased at 38 h after ischemia; the inactivation curves of I(A) shifted toward the depolarizing direction accordingly at 38 h after ischemia. The I(A) remained unchanged in CA3 neurons after ischemia. The amplitudes of delayed rectifier potassium currents (I(Kd)) in CA1 neurons were progressively increased after ischemia. No significant difference in I(Kd) was detected in CA3 and granule cells at any time points after reperfusion. These results indicated that the voltage dependent potassium currents in hippocampal neurons were differentially altered after cerebral ischemia. The up-regulation of I(A) in dentate granule cells might have protective effects. The increase of I(Kd) in CA1 neurons might be associated with the neuronal damage after ischemia.     

Bcl-2 and Bax proteins are increased in neocortical but not in thalamic apoptosis following devascularizing lesion of the cerebral cortex in the rat: an immunohistochemical study

The hypothesis that devascularization of somatosensory and motor cortex causes apoptosis in infarcted regions and in the linked thalamic nuclei was evaluated. To unravel whether Bcl-related proteins, known to regulate apoptosis, participate in neuronal and glial responses to devascularization, we analyzed immunohistochemically the distribution and intensity of staining of Bcl-2 and Bax proteins at different time points after lesion. Both early (up to 6 h) and late (1-7 days) responses were studied. Devascularization led to rapid (within hours) apoptosis in the cortex and to a delayed (within 3-7 days) apoptosis in thalamic nuclei. In control groups, Bcl-2 and Bax immunoreactivity (IR) was detected in neurons and oligodendrocytes but not in astrocytes or microglia. Following devascularization, Bcl-2 IR and Bax IR increased in neurons before the onset of the apoptosis. In the ischemic focus, the increase reached maximal values 3 h after the lesion. The increase was of slower onset in the penumbra zone (24 h and after), a region in which both proteins were induced in astrocytes also. The change of Bax IR intensity exceeded four times that of Bcl-2 at all time points investigated, indicating a diminution of Bcl-2/Bax ratio that may direct neurons to apoptotic pathway. In numerous neurons, an increase of IR in the cytoplasm was accompanied by induction of nuclear staining. No changes of Bcl-2 and Bax IR were found in thalamic nuclei. Our results point to different mechanisms underlying apoptosis of cortical and thalamic neurons. Nuclear appearance of Bcl-2 and Bax suggests they possess regulatory role of gene expression changes triggered by cortical infarct.     

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.     

Expression and changes of Ca2+-ATPase in neurons and astrocytes in the gerbil hippocampus after transient forebrain ischemia

Ca2+-ATPase is one of the most powerful modulators of intracellular calcium levels. In this study, we focused on chronological changes in the immunoreactivity and protein levels of Ca2+-ATPase in the hippocampus after 5 min of transient forebrain ischemia. Ca2+-ATPase immunoreactivity was significantly altered in the hippocampal CA1 region and in the dentate gyrus, but not in the CA2/3 region after ischemic insult. In the sham-operated group, Ca2+-ATPase immunoreactivity was detected in the hippocampus. Ca2+-ATPase immunoreactivity in the CA1 region and in the dentate gyrus, and its protein levels peaked 3 h after ischemic insult. At this time, CA1 pyramidal cells and dentate polymorphic cells showed strong Ca2+-ATPase immunoreactivity. Thereafter, Ca2+-ATPase immunoreactivity reduced in the CA1 region and in the dentate gyrus. One day after ischemic insult, Ca2+-ATPase immunoreactivity was observed in some CA1 non-pyramidal cells, and 4 days after ischemic insult, Ca2+-ATPase immunoreactivity was detected in astrocytes throughout the CA1 region, but Ca2+-ATPase immunoreactivity in the dentate gyrus had nearly disappeared. Our results suggest that Ca2+-ATPase changes may be associated with a response to ischemic damage in hippocampal CA1 pyramidal cells, and that increased Ca2+-ATPase immunoreactivity in the reactive astrocytes may be associated with the maintenance of intracellular calcium levels.     

Downregulation of COX-2 and JNK expression after induction of ischemic tolerance in the gerbil brain

The response of the inducible isoform of the prostaglandin H2 synthase (COX-2) and the c-Jun N-terminal kinase (JNK) in post-ischemic neuronal damage was assessed in a model of ischemic tolerance in Mongolian Gerbils. After a single 6-min bilateral carotid occlusion, histological damage was evident in the CA1 region of hippocampus, correlated with a high expression of JNK and COX-2 mRNA. However, in the group of animals with a 2-min ischemia and the tolerance group, in which a 2-min bilateral carotid occlusion was followed 3 days later by a 6-min ischemia, no hippocampal or cortical damage was detected. Accordingly, the JNK and COX-2 mRNA levels remained unaffected. We suggest that the level of JNK and COX-2 expression may determine the outcome as either post-ischemic cell death or tolerance.     

Reproducible loss of CA1 neurons following carotid artery occlusion combined with halothane-induced hypotension

The 2-vessel occlusion approach to produce global ischemia in rats requires concomitant reduction of systemic blood pressure. We have utilized the hypotensive effect of halothane administrated by artificial respiration to prevent respiratory arrest and to ensure stable physiological conditions. Systemic blood pressure was reduced to 40-45 mmHg by instant adjustments of the halothane concentration. Bilateral occlusion of the carotid arteries caused a profound and reproducible ischemia, as analyzed by laser-Doppler flowmetry. In the rats exposed to 11, 12, or 13 min of ischemia, 5% died and 5% developed seizures. The extent of neuronal death in CA1 was highly correlated to the duration of ischemia. Following 11 min of ischemia, CA1 neuronal cell death, as analyzed by Fluoro-Jade, was absent 1 day after injury, variable at day 4, and consistent at day 7. The numbers of cresyl violet- and NeuN-positive neurons at day 7 were 8% and 20% of control, respectively. OX42 immunoreactivity was low and variable at day 4, but pronounced at day 7. In conclusion, this rat global ischemia model is relatively simple to perform, has a low mortality, and produces a profound and highly reproducible delayed cell death of hippocampal CA1 neurons.     

Adenine nucleotide metabolism and cell fate after oxidant exposure of rat cortical neurons: effects of inhibition of poly(ADP-ribose) polymerase

We exposed cultured neurons prelabeled with 14C-adenine to H2O2 with or without the poly(ADP-ribose) polymerase (PARP) inhibitor 3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) to quantify its effects on acute ATP depletion, later ATP synthesis, cellular and nuclear morphology, extent of DNA fragmentation, and PARP cleavage. According to the extent of the acute ATP depletion, the exposures were classified as 'mild' (50 microM H2O2), 'moderate' (100-250 microM H2O2), or 'severe' (500 microM-1 mM H2O2) insults. Mild exposure had no significant effects on the parameters studied. In the 'moderately' exposed neurons, ATP depletion to 59+/-6% of control was associated with a decrease in the cell counts, apoptotic morphology, and cleavage of PARP. In this group, DPQ prevented the acute ATP (to 95+/-15% of control), preserved cell morphology, and improved cell survival. In the 'severe' group, ATP depletion to 18+/-4% was associated with necrosis and intact PARP. DPQ elevated ATP levels (to 44+/-12% of control) and post-insult ATP synthesis, improved cell counts, and altered cell morphology towards apoptosis rather than necrosis. Post-insult application of DPQ was less effective. Our results show that the extent of oxidant-induced ATP depletion and cell fate can be modified by PARP inhibition, to some extent also after the insult.     

Changes in number of synapses and mitochondria in presynaptic terminals in the dentate gyrus following cerebral ischemia and rehabilitation training

Damage to the adult brain can result in adaptive plasticity in regions adjacent to the site of the principal insult and that the plastic changes may be modulated by post-injury rehabilitation training. In this study, we examined the effects of rehabilitation training on synaptic morphology in the dentate gyrus following transient global cerebral ischemia and the metabolic correlates of the ultrastructural changes. Forty adult male Wistar rats were included in the study and assigned to either ischemia or sham group. Following ischemic or sham surgery, rats were randomized to either complex environment housing (EC), exercise (EX), or social condition (SC, paired housing) group. Electron microscopy and unbiased stereological methods were used to evaluate synaptic plasticity and the number and size of mitochondria in synaptic axon terminals. Increased number of granule neurons was seen in all ischemic groups and in the sham EC rats. Changes in the number of synapses per neuron in the outer and inner molecular layers of the dentate gyrus parallel those seen in granule neurons. Similarly, ischemia and behavioral experience in EC independently increased the number of synaptic mitochondria in presynaptic terminals in both the outer and inner molecular layers; however, no significant changes were seen in mitochondrial size. These data suggest a link between behavioral training and synaptic plasticity in the region adjacent to the injury and that the likely metabolic correlate of this synaptic plasticity is increased number of mitochondria at synaptic axon terminals.     

Sevoflurane-induced preconditioning of rat brain in vitro and the role of KATP channels

In the present study we tested the ability of the inhalation anesthetic sevoflurane to induce preconditioning against hypoxia in vitro. Rat hippocampal slices were prepared using established procedures. After 90 min of incubation, slices were exposed for 30 min to 0, 1, 2 or 3 minimum alveolar concentration (MAC) of sevoflurane under normoxic conditions (95% O2/5% CO2). Fifteen minutes later, slices were exposed to 13-min hypoxia (95% N2/5% CO2) followed by 30-min reoxygenation. The amplitude of extracellularly recorded, orthodromically evoked, CA1 population spikes (neuronal function) at the end of the reoxygenation period was measured and used to quantify the degree of recovery of neuronal function posthypoxia. To assess the role that the mitochondrial KATP channel plays in preconditioning, its blocker, 5-hydroxydecanoic acid (5-HD), was added during sevoflurane exposure. Sevoflurane-preconditioning with 1, 2 and 3 MAC increased the degree of recovery of neuronal function after 13-min hypoxia and 30-min reoxygenation from 51 +/- 1% (0 MAC), to 55 +/- 3%, 63 +/- 3%, and 72 +/- 2%, respectively. The effect of 3 MAC sevoflurane was blocked by 5-HD (53 +/- 3%), whereas 5-HD alone had no effect (48 +/- 3%) on the recovery of neuronal function from hypoxia. It is concluded that sevoflurane is capable of inducing preconditioning in vitro in a dose-dependent fashion and involves activation of mitochondrial KATP channels.     

Acute changes in cortical excitability in the cortex contralateral to focal intracerebral hemorrhage in the swine

Injury to the cerebral cortex results in functional deficits not only within the vicinity of the lesion but also in remote brain regions sharing neuronal connections with the injured site. To understand the electrophysiological basis of this phenomenon, we evaluated the effects of a focal intracerebral hemorrhage (ICH) on cortical excitability in a remote, functionally connected brain region. Cortical excitability was assessed by measuring the somatic evoked potential (SEP) elicited by electrical stimulation of the swine snout, which is somatotopically represented in the rostrum area of the primary somatosensory (SI) cortex. The SEP was measured on the SI cortex ipsilateral to the site of ICH and on the contralateral SI cortex during the acute period (< or =11 h) after collagenase-induced ICH. The ICH rapidly attenuated the SEP on the ipsilateral cortex as we reported earlier. Interestingly, the ICH also attenuated the SEP on the contralateral SI cortex. Evoked potentials in the contralateral SI cortex showed a gradual decrease in amplitude during this acute period of ICH. We then investigated whether the interhemispheric connections shared by the contralateral SI and the lesion cortex were responsible for the diminished evoked potentials in the uninjured hemisphere after ICH. A separate group of animals underwent corpus callosal transection prior to electrocorticography (ECoG) recordings and ICH injury. Within hours of hemorrhagic injury, a gradual but marked increase in evoked potential amplitude was observed in the homotopic SI cortex of callosotomized animals as compared to pre-injection recordings. The enhancement suggests that there are additional effects of ICH on remote areas functionally connected to the site of injury. Functional deficits were present in both SI cortices within the first several hours of a unilateral injury indicating that the cessation of brain activity in the lesioned SI is mirrored in the contralateral hemisphere. This electrophysiological depression in the uninjured SI cortex is mediated in part by the interhemispheric connections of the corpus callosum.     

Altered production of nitric oxide and reactive oxygen species in rat nodose ganglion neurons during acute hypoxia

Nitric oxide (NO) production in the sensory neurons of the rad nodose ganglion was studied by examining the distribuiotn of NO synthase (NOS) by use of NADPH diaphorase (NADPHD) histochemistry and immunohistochemistry ofr the presence of isoformes of NOS: neuronal (nNOS), endothelial (eNOS) and the inducible isoform (iNOS). Distribution and changes in NO production during acute hypoxia were studied in vital vibratome sections with the fluorescent marker for NO, diaminotriazolofluorescein (DAF-2T). Furthermore, changes in reactive oxygen species (ROS) in vibratome slices were examined utilizing 2',7'-dichlorofluorescein (DCF). By use of these histochemical methods, a positive NADPH reaction and positive immunoreactivity for eNOS were noted in all neurons observed. While for nNOS immunoreactivity, both strongly positive cells but also many negative cells are seen., no iNOS immunoreactive cells were observed. In vital vibratome slices, a dot-like distribution of fluorescence for DAF-2T, indicating production of NO, was observed in the nodose ganglion cells. Neurons exposed to hypoxia showed stronger DAF-2T fluorescence than cells exposed to normoxia, indicating an increased production of NO during hypoxia. When Ca(2+) was removed from the incubation buffer, the intensity of fluorescence for DAF-2T decreased but did not disappear completely. Using a photoconversion technique, DAF-2T was localized in the inner membrane of mitochondria in the ganglion cells by electron microscopy. The level of DCF signals for detection of ROS was higher in neurons incubated in the normoxic medium than those incubated under conditions of hypoxia. Nerve cells exposed to hypoxia followed by reoxygenation (3 min in normoxic conditions) showed higher fluorescence for DCF than those exposed to normoxia. The results of the present study demonstrate clearly that the basal production of NO in viscerosensory neurons is increased during hypoxia and is due to the isoform eNOS rather than nNOS, moreover, that ROS is augmented by reoxygenation but not during hypoxia.     

Calbindin D-28k is expressed in the microvascular basal lamina in the ventral horn at early time after transient spinal cord ischemia in the rabbit

Much evidence has been accumulated that the increased expression of calbindin D-28k (CB) is involved in the blockade of calcium-evoked excitotoxicity in cerebral ischemia. We investigated the expression of CB in the basal lamina of microvessels in the ventral horn of the rabbit spinal cord after transient spinal cord ischemia. Spinal cord sections at the level of L7 were immunostained using monoclonal antibody raised against CB at light and electron microscopic levels. CB immunoreactivity was detected in the basal lamina of microvessels at 30 min after ischemic insult. By 3 h after ischemia, CB immunoreactivity was increased in the basal lamina of the microvessels. CB immunoreactivity began to decrease at 6 h after ischemia and nearly disappeared at 48 h after ischemic insult. For calcium detection in the blood vessels of spinal cord, we conducted an alizarin red staining. Alizarin red reactivity was detected in some microvessels at 3 h after ischemic insult. Our results suggest that the ectopic expression of CB in the microvascular basal laminae may be associated with the buffering of calcium in the endothelial cells of microvessels after ischemic damage.     

Temporary focal ischemia in the mouse: technical aspects and patterns of Fluoro-Jade evident neurodegeneration

Animal models of cerebral infarction are crucial to understanding the mechanisms of neuronal survival following ischemic brain injury and to the development of therapeutic interventions for victims of all types of stroke. Rodents have been used extensively in such research. One rodent model of stroke utilizes either permanent or temporary occlusion of the middle cerebral artery (MCAO) to produce ischemia. Since the development of an endovascular method for this was published in 1989, MCAO has been applied commonly to the rat, and often paired with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining for stroke volume measurement. Meanwhile, advances in the ability to genetically alter mice have allowed exciting lines of research into ischemia. Because of technical demands and issues with survival, relatively few laboratories have investigated the MCAO method in the mouse. Our present work utilizes a mouse middle cerebral occlusion (MCAO) model of embolic stroke to study neuronal degeneration following temporary focal cerebral ischemia. C57Bl/6J mice were used to examine the exact effects of MCAO using Fluoro-Jade, a marker of neurodegeneration that allows observation of specific brain regions and cells destined to die. A time course of escalating neuronal degeneration from 10 min to 7 days following MCAO was established. Technical aspects of this popular method for transient focal ischemia as it applies to the mouse are discussed.     

Ischemic intensity influences the distribution of delayed infarction and apoptotic cell death following transient focal cerebral ischemia in rats

The aim of this study was to investigate whether the apoptotic process contributes to the delayed infarction that follows a middle cerebral artery (MCA) occlusion of 20 min (mild ischemia group) and to compare this with the delayed component of infarct following 2 h of MCA occlusion (severe ischemia group). Adult male Sprague-Dawley rats underwent left MCA occlusion for either 20 min or 2 h and were reperfused for 12, 24 and 72 h. On 2,3,5-triphenyltetrazolium chloride-stained coronal sections, delayed infarction was observed to develop in the whole MCA territory after mild ischemia, and also in the frontoparietal cortex after severe ischemia. At 24 h after 20 min of MCA occlusion, characteristic apoptotic features, including chromatin condensation and apoptotic bodies were frequently observed by electron microscopy. In both ischemic groups, Hoechst 33342 staining showed typically condensed and fragmented nuclei in the area showing delayed infarction, where TdT-dUTP nick end labeling (TUNEL)-positive cells were also significantly increased. Caspase-3 activity was also found to be elevated 24 and 72 h after reperfusion and this peaked at 24 h in both groups. These findings suggest that ischemic severity may influence the distribution of delayed infarction, and that apoptosis is the underlying pathophysiologic mechanism.     

IGF-1 protects oligodendrocyte progenitor cells and improves neurological functions following cerebral hypoxia-ischemia in the neonatal rat

To investigate if insulin-like growth factor-1 (IGF-1) provides neuroprotection to oligodendrocyte progenitor cells (OPCs) following cerebral hypoxia-ischemia, a previously developed neonatal rat model of white matter damage was used in this study. Postnatal day 4 (P4) SD rat pups were subjected to bilateral common carotid artery ligation, followed by exposure to 8% oxygen for 10 min. IGF-1 (0.5 microg) or vehicle was injected into the left ventricle after artery ligation and before the hypoxic exposure. Cerebral hypoxia-ischemia caused death of O4+ late OPCs in the P5 rat brain and impaired myelination in the P9 and P21 rat brain. Caspase-3 activation was involved in the death of OPCs. Moreover, cerebral hypoxia-ischemia impaired neurobehavioral performance in juvenile rats. IGF-1 treatment attenuated damages to OPCs and improved neurological functions after cerebral hypoxia-ischemia. It reduced death of O4+ OPCs by 39% on P5 and enhanced myelination on P9 and P21. Bromodeoxyuridine uptake assay showed that cerebral hypoxia-ischemia inhibited proliferation of stem/progenitor cells in the subventricular zone and NG2+ early OPCs in the white matter area. IGF-1 treatment increased cell proliferation in the subventricular zone by 31% 1 day following hypoxic-ischemic insult. Proliferation of early and late OPCs in the IGF-1-treated group was 1.5- and 2.4-fold of that in the vehicle-treated group, respectively. In conclusion, IGF-1 provided potent neuroprotection to OPCs and improved neurological functions following cerebral hypoxia-ischemia in the neonatal rat. The neuroprotection of IGF-1 was associated with its antiapoptotic and mitogenic effects.     

Behaviorally-induced ultrastructural plasticity in the hippocampal region after cerebral ischemia

Behavioral training has been shown to induce synaptic plasticity in both intact and injured animals. Because of the possibility that the adaptive changes after ischemic damage may make the brain more malleable to behavioral training, we examined the effects of complex environment (EC) housing and exercise (EX) after global cerebral ischemia on synaptic structural alterations. Forty-two adult male Wistar rats were included in the study and assigned to either ischemia or sham group. Following ischemic or sham surgery, rats were randomized to either EC, EX, or social condition (SC, paired housing) group. CA1 was processed for electron microscopy and unbiased stereological techniques were used to evaluate plasticity. Significantly decreased neuron density was seen in anterior and medial CA1 in ischemic animals regardless of behavioral training. Neuron density in anterior CA1 was 31% less than the medial area. Synaptogenesis was influenced by cerebral ischemia and behavioral training in that all ischemic groups and sham EC animals showed greater number of synapses per neuron compared to the sham EX and SC groups. Analysis of synapse configuration showed that the synaptogenesis in ischemia EX and SC rats was formed mainly by synapses with single synaptic boutons, whereas in the ischemia EC and sham EC rats synaptogenesis was formed mainly by synapses with multiple synaptic boutons. Furthermore, housing of sham and ischemia rats in EC resulted in increased number of synapses with perforated postsynaptic density. Together, these data suggest that behavioral experience in EC after insult may be able to enhance synaptic plasticity.     

Mild hypothermia enhances the neuroprotective effects of FK506 and expands its therapeutic window following transient focal ischemia in rats

FK506 (tacrolimus), an immunosuppressant, reportedly reduces ischemic brain injury following transient middle cerebral artery occlusion (MCAO) in rats. The authors previously reported that the therapeutic window of FK506 in this model is more than 1 h, but less than 2 h. The aim of the present study is to determine whether mild hypothermia (35 degrees C) enhances the neuroprotective effects of FK506 and expands its therapeutic window. Sprague-Dawley rats were subjected to 2 h MCAO followed by 24 h reperfusion. Animals were randomly divided into four groups: (I) vehicle-treated normothermic group; (II) FK506-treated normothermic group; (III) vehicle-treated hypothermic group; (IV) FK506-treated hypothermic group. Animals received a single injection of FK506 (0.3 mg/kg) or vehicle intravenously at 2 h after ischemic induction. During ischemia, temporal muscle and rectal temperatures were maintained at 37 degrees C in the normothermic animals and at 35 degrees C in the hypothermic animals. Infarct volumes and neurological performance were evaluated at 24 h after reperfusion. The combination of FK506 and mild hypothermia significantly reduced infarct volume (cortex, -61%; striatum, -31%) and edema volume (cortex, -57%; striatum, -41%), while mild hypothermia or FK506 alone failed to improve ischemic brain damage. Furthermore, this combination also provided for the best functional outcome. These results demonstrate that the combination of FK506 and mild hypothermia significantly reduces ischemic brain damage following transient MCAO in rats, and expands the therapeutic window for FK506. This therapy may be a new approach for treatment of acute stroke.     

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.     

Transient forebrain ischemia effects interaction of Src, FAK, and PYK2 with the NR2B subunit of N-methyl-D-aspartate receptor in gerbil hippocampus

Two different models of brain ischemia were used to examine the evoked changes in the tyrosine phosphorylation of NMDA receptor subunits 2A and 2B (NR2A and NR2B), as well as their interactions with non-receptor tyrosine kinases (NRTKs: FAK, PYK2 Src), and PSD-95 protein. Only short-term 5 min ischemia followed by 3 h reperfusion resulted in the elevated tyrosine phosphorylation of both investigated NMDA receptor subunits, but in contrast to previously published data, more pronounced in the case of NR2B. Concomitantly, an increased association of NR2B with FAK, PYK2, Src and PSD-95 has been observed. This sharp early reaction to brief ischemia was markedly attenuated during prolonged recovery (72 h) with almost complete return to control values. The initial recruitment of tyrosine kinases to NMDA receptor during the first 3 h of reperfusion is generally consistent with an active postischemic remodeling of PSD and may participate in the induction of the postischemic signal transduction pathway in gerbil hippocampus. In contrast, ischemia of longer duration (up to 30 min) caused an immediate decrease in the protein levels as well as tyrosine phosphorylation of both NR2A and NR2B subunits which was accompanied by the marked attenuation of the association with their investigated molecular partners--PSD-95 and NRTKs. This effect may be mimicked in vitro by Ca2+-dependent activation of endogenous calpains in purified PSD preparation suggesting irreversible deterioration of the synaptic signaling machinery during irreversible long-term ischemia.