Min Of Transient Cerebral Ischemia Research Articles

Changes in the expression of mitochondrial peroxiredoxin and thioredoxin in neurons and glia and their protective effects in experimental cerebral ischemic damage

We observed chronological changes in the mitochondrial-specific antioxidant enzymes peroxiredoxin 3 (Prx3) and thioredoxin 2 (Trx2) and their neuroprotective effects in the hippocampal CA1 region after 5 min of transient cerebral ischemia in gerbils. In the sham-operated group, weak Prx3 and Trx2 immunoreactivity was detected in the stratum pyramidale. Prx3 immunoreactivity was increased in pyramidal neurons and expressed in microglia 1 and 3 days, respectively, after ischemia/reperfusion (I/R). Trx2 immunoreactivity in pyramidal neurons increased 30 min and 1 day after I/R and decreased 6 h after I/R. Trx2 immunoreaction was expressed in astrocytes at 3 days postischemia. The intraventricular administration of Prx3 or Prx3/Trx2 (16 μg/20 μl, icv) using an osmotic pump significantly reduced ischemia-induced hyperactivity in a spontaneous motor test and protected CA1 pyramidal neurons from the ischemic damage. In addition, the activation of astrocytes and microglia was decreased in the ischemic CA1 region after Prx3/Trx2 treatment. In addition, treatment with Prx3 or Prx3/Trx2 significantly reduced lipid peroxidation and the release of cytochrome c from mitochondria and cytoplasm in the ischemic CA1 region. These results suggest that changes in the expression of Prx3 and Trx2 in the hippocampal CA1 region after I/R may be associated with the delayed neuronal death of CA1 pyramidal cells induced by transient cerebral ischemia, and that treatment with Prx3 or Prx3/Trx2 in ischemic brains shows a potent neuroprotective effect against ischemic damage by reducing lipid peroxidation and mitochondrial-mediated apoptosis by I/R.

Doublecortin-immunoreactive Neuroblasts in Each Layer of the Main Olfactory Bulb After Transient Cerebral Ischemia in Gerbils

Neurogenesis in the adult brain occurs continuously throughout life. The main olfactory bulb (MOB) is the first central relay of the olfactory system. We examined proliferation of newly generated cells in each layer of the gerbil MOB after 5 min of transient cerebral ischemia using doublecortin (DCX), a marker of neuronal progenitors. Many DCX immunoreactive neuroblasts were found in the all layers of the MOBs of control and ischemia groups. Ten to 15 days after ischemia/reperfusion, no difference in numbers of DCX immunoreactive neuroblasts was found in the MOB. Thirty days after ischemia/reperfusion, significant increase of DCX immunoreactive cells was observed in all layers of ischemic MOB. This result indicates that neuroblasts increase in the MOB from 30 days after transient cerebral ischemia in gerbils.

An ultrastructural analysis of cellular death in the CA1 field in the rat hippocampus after transient forebrain ischemia followed by 2, 4 and 10 days of reperfusion

An ultrastructural study was performed to investigate the type of cellular death that occurs in hippocampal CA1 field pyramidal neurons after 10 and 20 min of transient cerebral ischemia in the male adult Wistar rats, followed by 2, 4 and 10 days of reperfusion. The four-vessel occlusion method was used to induce ischemic insult for either 10 or 20 min, following which the animals were submitted to either 2, 4 or 10 days of reperfusion. The animals were then anaesthetised, and their brains removed, dehydrated, embedded, sectioned and examined under a transmission electron microscope. After ischemic insult, neurons from the CA1 field presented alterations, corresponding to the initial, intermediate and final stages of the degenerative process. The only difference observed between the 10 and 20 min ischemic groups was the degree of damage; the reaction was stronger in 20 min groups than in the 10 min groups. While neurons were found in the different stages of oncotic necrosis in all groups, differences were found between the groups in relation to prevalent stages. In both ischemic groups, after 2 days of reperfusion, the initial stage of oncotic necrosis was prevalent and large numbers of neurons appeared normal. In both groups, after 4 days of reperfusion, most of the neurons showed more advanced alterations, typical of an intermediate stage. In both groups, after 10 days of reperfusion, alterations corresponding to the intermediate and final stages of oncotic necrosis were also predominant. However, few intact neurons were identified and the neuropile appeared more organised, with numerous glial cells. In summary, the pyramidal neurons of the CA1 field displayed selective vulnerability and exhibited a morphological death pattern corresponding exclusively to an oncotic necrotic pathway.

Immunohistochemical Detection of Apoptosis-Related Proteins in Gerbil Hippocampus Transient Cerebral Ischemia: Neuroprotective Effect of Pitavastatin

Delayed and selective neuronal damage was caused in the CA1 sector of hippocampus following 5 min of transient cerebral ischemia in gerbils. We investigated the immunohistochemical alterations of apoptosis-related proteins such as bcl-2α, bcl-xs/l, bax, cytochrome c, and active caspase 3 and TUNEL staining in the hippocampus at 1 and 5 hr and 1, 2, 5 and 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the alterations of apoptosis-related proteins and TUNEL staining in the hippocampus after cerebral ischemia. The alterations of apoptosis-related proteins in the hippocampal CA1 sector were more pronounced than the changes of hippocampal CA3 sector and dentate gyrus after cerebral ischemia. The alterations of apoptosis-related proteins in the hippocampal CA1 sector after cerebral ischemia preceded the neuronal damage in this region. Furthermore, the study with TUNEL staining showed that a marked increase of TUNEL-positive nuclei was evident only in the hippocampal CA1 sector 5 days after cerebral ischemia. Our immunohistochemical study also showed that pitavastatin prevented the alterations of apoptosis-related proteins and the increase of TUNEL-positive nuclei in the hippocampal CA1 sector 5 days after cerebral ischemia. The present study indicates that transient cerebral ischemia in gerbils causes the mitochondrial-dependent apoptosis in the hippocampal CA1 sector. Furthermore, our present study demonstrates that pitavastatin can prevent the alterations of apoptosis-related proteins and the increase of TUNEL-positive nuclei in the hippocampal CA1 sector after cerebral ischemia. Thus our study provides novel therapeutic strategies in clinical stroke.

Nitric oxide synthase inhibition and delayed cerebral injury after severe cerebral ischemia in fetal sheep.

After transient cerebral ischemia in fetal sheep, delayed disruptions in cerebral energetics are represented by a delayed increase in cortical impedance, a progressive decrease in the concentration of oxidized cytochrome oxidase as measured by near-infrared spectroscopy, and cortical seizures. Because the production of nitric oxide (NO), a potent mediator of neuronal death, is increased during this phase, the present study investigated whether inhibition of NO synthesis could ameliorate the delayed disruption in cerebral energetics. Eleven late gestation fetal sheep were subjected to 30 min of transient cerebral ischemia in utero. Two hours later, the treatment group (n = 5) received a continuous infusion of N(G)-nitro-L-arginine, a competitive inhibitor of NO synthase, whereas the control group (n = 6) received PBS. Changes in concentration of oxidized cytochrome oxidase, cortical impedance, and electrocortical activity were observed for 3 d. A delayed increase in cortical impedance of similar magnitude and duration commenced at 14+/-4 h in the control and at 15+/-3 h in the treatment groups. The progressive decrease in oxidized cytochrome oxidase signal, by -2.2+/-0.2 micromol/L in the control and -2.0+/-0.4 micromol/L in the treatment group at 72 h postischemia, was similar in both groups. In both groups, delayed cortical seizures were indicated by intense low-frequency electrocortical activity. In the treatment group, duration of cortical seizures was increased and the intensity of the final electrocortical activity was more depressed (-19+/-1 dB versus -10+/-2 dB). The results indicate that after cerebral ischemia in fetal sheep, NO synthase inhibition does not ameliorate the delayed disruptions in cerebral energetics. However, the effect of NO synthase inhibition on delayed cortical seizures may improve our understanding of the role of NO during this phase.

Delayed recovery of auditory cortical evoked potentials is correlated with cortical neuronal death after transient cerebral ischemia in awake gerbils

Early detection of irreversible neuronal change after transient cerebral ischemia is important so that adequate treatment can be initiated within the therapeutic window. We have examined the correlation between changes in middle-latency auditory evoked potentials (MAEPs) and histological changes in the auditory cortex of awake Mongolian gerbils subjected to 4 min or 12 min of transient cerebral ischemia. Post-ischemic MAEPs were characterized by the appearance of a markedly large negative and positive component at approximately 17–22 ms latency in both groups. Delay in the appearance of the high amplitude (maximal amplitude at 45 min after recirculation in the 12-min ischemia group) precedes the slowly developing death of neurons in the auditory cortex that results from transient cerebral ischemia.

Postischemic changes of

Sequential alterations of [3H]nimodipine and [3H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [3H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [3H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [3H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [3H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [3H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [3H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [3H]nimodipine and [3H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage. Copyright Rapid Science Ltd

Effect of hyperglycemia on reperfusion-associated recovery of intracellular pH and high energy phosphates after transient cerebral ischemia in gerbils.

Hyperglycemia increases cerebral damage after transient cerebral ischemia. This study used in vivo 31P nuclear magnetic resonance spectroscopy to determine the relationship of intracellular tissue acidosis and delayed recovery of brain high-energy phosphates to increased damage during the reperfusion period. Mongolian gerbils were subjected to transient bilateral carotid ischemia for 20 min with 2 h reperfusion. All gerbils were pretreated intraperitoneally with equivalent volumes in saline of 0.003 units per kilogram of insulin or vehicle, or with 4 grams of glucose per kilogram. The gerbils were then scanned in a 4.7 Tesla Magnetic Resonance Imager-Spectrometer to determine levels of intracellular pH, inorganic phosphate, adenosine triphosphate, and phosphocreatine. In each group, intracellular pH decreased with ischemia, but most significantly in hyperglycemic animals (6.45 +/- 0.15), in which it had not recovered to preischemic levels by the end of the reperfusion period (6.8 +/- 0.1 vs 7.04 +/- 0.1, p < 0.05). High-energy phosphates phosphocreatine-inorganic phosphate and phosphocreatine-adenosine triphosphate showed partial recovery in all groups throughout the reperfusion period; the recovery was not significantly altered by glucose status. Hyperglycemia worsened pH but not the recovery of high-energy phosphates in animals reperfused after 20 min of transient cerebral ischemia. This sustained acidosis may be a primary event in transient damage in hyperglycemic animals.

Nitric oxide synthase inhibition attenuates delayed vasodilation and increases injury after cerebral ischemia in fetal sheep.

Transient cerebral ischemia in fetal sheep is followed by a period of delayed cerebral injury associated with cerebral vasodilation. As nitric oxide (NO) can mediate both vasodilation and neuronal death, this study investigated whether inhibition of NO synthesis would attenuate the vasodilation and decrease cerebral injury. Eleven late gestation (range 122-133 d) fetal sheep were subjected to 30 min of transient cerebral ischemia in utero. Two hours later, treatment group (n = 5) received a continuous infusion of NG-nitro-L-arginine (L-NNA) at a dose of 50 mg.h-1 for 4 h followed by 20 mg.h-1 for the subsequent study period, a competitive inhibitor of NO synthase (NOS), whereas a control group (n = 6) received PBS. Inhibition of NOS activity was confirmed in the treatment group by 1) suppression of the fall in mean arterial blood pressure (MAP) associated with acetylcholine (p < 0.01), and 2) persistent increase in MAP after commencement of L-NNA (p < 0.05). Changes in cerebral blood volume (CBV) were observed for 3 d by measuring changes in concentration of total cerebral Hb ([tHb]) using near infrared spectroscopy. The delayed increase in CBV commenced at 13.1 +/- 1.0 h postischemia in the control and 12.7 +/- 2.3 h in the treatment group. Maximum increase at 30-36 h was 0.5 +/- 0.1 mL.100 g-1 in the treatment group and 1.2 +/- 0.2 mL.100 g-1 in the control (p < 0.05). Final CBV was depressed below preischemic baseline in the treatment (-0.7 +/- 0.2 mL.100 g-1) but not the control group (-0.1 +/- 0.3 mL.100 g-1) (p < 0.05). Neuronal loss, quantified histologically 3 d postischemia, indicated that cerebral injury was increased in the treatment group (p < 0.05). The results indicate that after transient cerebral ischemia in fetal sheep, NOS inhibition attenuates the delayed rise in CBV but does not decrease the extent of cerebral injury.

Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia.

Intra-ischemic moderate hypothermia generally protects the brain against ischemic cell death, while hypothermia instigated several hours into the reperfusion phase is considered to be less effective. Here we report the effect of hypothermia (32.5 degrees - 33.5 degrees C) of 5-h duration, initiated at 2, 6, 12, 24 and 36 h into the recirculation phase following 10 min of transient cerebral ischemia, on ischemic neuronal injury in the hippocampus and striatum of the rat. Hypothermia induced at 2 h, and 6 h postischemia reduces neuronal damage in the entire hippocampal CA1 region by approximately 50%. In the lateral CA1 region hypothermia induced at 12 h postischemia, significantly mitigates necrosis. When initiated at 2 h postischemia, but not later, protection was also observed in the striatum. Hypothermia induced 24 and 36 h postischemia was ineffective. A period of hypothermia of 5 h, initiated 2 h postischemia, was required for marked neuronal protection in the CA1 region, while 3.5-h hypothermia decreased neuronal damage by approximately 10% and 30 min hypothermia was ineffective. The clinical implications of the data are that extended period of hypothermia initiated long into the recovery phase following ischemia may prove beneficial. Hypothermia protects brain regions displaying rapid as well as delayed neuronal damage, and a minimal time of hypothermia is required for effective neuronal protection. Also, strict temperature control for up to 24 h postischemia may be required for proper assessment of the efficacy of cerebro-protective drugs.

Postischemic changes in the binding of excitatory and inhibitory neurotransmitters in the gerbil brain

We performed receptor autoradiography to determine sequential changes in the binding of N- methyl- D - aspartate (NMDA) and γ-aminobutyric acid A (GABA A) 1 h to 1 month after 10 min of transient cerebral ischemia in the gerbil. [ 3H]MK-801 and [ 3H]muscimol were used to label NMDA and GABA A receptors, respectively. [ 3H]MK-801 binding showed no significant changes in the striatum and hippocampus at an early stage (1–24 h) after ischemia. Thereafter, [ 3H]MK-801 binding exhibited a significant reduction in the dorsolateral striatum, most of hippocampal CA1 sector and dentate gyrus 48 h or 7 days of recirculation. However, [ 3H]MK-801 binding progressively depressed in the hippocampal CA1 sector 1 month after ischemia, whereas other regions showed no significant alteration in the binding. By contrast, [ 3H]muscimol binding was unchanged in all brain areas throughout the recirculation period. A histological study also demonstrated that transient ischemia caused severe neuronal damage in the striatum and hippocampus. These results demonstrate that NMDA and GABA A receptors are relatively resistant to severe degenerative processes. Furthermore, our finding suggests that transient ischemia may induce long-term changes in the properties of survival neurons or interneurons especially in the hippocampal CA1 sector.

Postischemic blockade of AMPA but not NMDA receptors mitigates neuronal damage in the rat brain following transient severe cerebral ischemia.

Glutamatergic transmission is an important factor in the development of neuronal death following transient cerebral ischemia. In this investigation the effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists on neuronal damage were studied in rats exposed to 10 min of transient cerebral ischemia induced by bilateral common carotid occlusion combined with hypotension. The animals were treated with a blocker of the ionotropic quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor, 2.3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), given postischemia as an intraperitoneal bolus dose of 30 mg kg-1 followed by an intravenous infusion of 75 micrograms min-1 for 6 h, or with the noncompetitive NMDA receptor blocker dizocilpine (MK-801) given 1 mg kg-1 i.p. at recirculation and 3 h postischemia, or with the competitive NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), 5 mg kg-1, given intraperitoneally at recirculation. Treatment with NBQX provided a significant reduction of neuronal damage in the hippocampal CA1 area by 44-69%, with the largest relative decrease in the temporal part of the hippocampus. In neocortex a significant decrease in the number of necrotic neurons was also noted. No protection could be seen following postischemic treatment with dizocilpine or CGP 40116. Our data demonstrate that AMPA but not NMDA receptor antagonists decrease neuronal damage following transient severe cerebral ischemia in the rat and that the protection by NBQX may be dependent on the severity of the ischemic insult. We propose that the AMPA receptor-mediated neurotoxicity could be due to ischemia-induced changes in the control mechanisms of AMPA receptor-coupled processes or to changes of AMPA receptor characteristics.

Decreases in excitatory synaptic transmission and increases in recurrent inhibition in the rat dentate gyrus after transient cerebral ischemia

Excitatory transmission along the perforant path from the entorhinal cortex to the granule cells of the dentate gyrus was evaluated two days after 10 min of transient cerebral ischemia in the rat. The amplitude of the population spike, and the amplitude and the initial slope of the population excitatory postsynaptic potential (EPSP) evoked by the perforant path stimulation were measured across a range of stimulus intensities, and were compared with control values. Inhibitory interactions were evaluated using the paradigm of paired pulse stimulation, comparing the amplitude of the population spike evoked by the second pulse of a pair to the initial spike. The maximal values of the initial slope of the population EPSP and the population spike were reduced in the ischemic group. Also, the extent of paired pulse inhibition was greater in the ischemic group. These results suggest that: (1) excitatory synaptic transmission along the perforant path is impaired in the postischemic period, (2) inhibition of the dentate granule cells is enhanced in this period. These results are not consistent with the hypothesis that there is a hyperactivation of the tri-synaptic circuit in the chronic postischemic period that accounts for the excitotoxic death of CA1 neurons.

Curative effect of an almitrine-raubasine combination in the postischemic syndrome following transient cerebral ischemia in dogs.

Cerebral hemodynamic and metabolic changes, occurring during delayed hypoperfusion following transient cerebral ischemia, and the influence of treatment with almitrine plus raubasine were studied in mongrel dogs. 10 min of transient cerebral ischemia was induced by bilateral clamping of both carotid and vertebral arteries. After declamping, the mean time necessary until cerebral venous PO2 (cvPO2) reached a value of 3.6 kPa, threshold for tissue hypoxia, was 80 min. At this time (T0), venous cerebral blood flow (vCBF) and cerebral perfusion pressure (Perf P) were below (60 and 20%, respectively) preischemic values, while cerebral vascular resistance (CVR) and oxygen and glucose extraction rates increased despite a normal cerebral oxygen consumption (CMRO2). At T0 ventilatory assistance without (control group) or with (treated group) intravenous infusion of almitrine plus raubasine was applied for 110 min. Between T0 and T110 min, 2 dogs died in the control group. During this period vCBF decreased by more than 60% in the control group while it slightly increased in the treated group. A strong decrease in Perf P (40%) and increase in CVR (140%) was observed in the control group while in the treated group Perf P and CVR slightly decreased (14 and 35%, respectively). CMRO2 decreased by 60% in the control group but remained within the normal range in the treated group. The fact that cvPO2 remained constantly below the initial value of 3.6 kPa in the control group and, on contrary, above this value in the group infused with raubasine plus almitrine indicates that the vCBF improvement leads to an increase in oxygen supply and is involved in the keeping of the adequacy between flow and metabolism. Our results support the hypothesis that the post-ischemic syndrome may play an important role in the acute prognosis of stroke. They clearly indicate that early cerebral resuscitation by infusion of almitrine plus raubasine, maintaining oxygen availability and CBF above initial thresholds, should improve the long-term neurological outcome.

Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat.

The aim of this morphological study was to determine the vulnerability of hippocampal interneurons to ischemia in the adult rat. Two types of interneurons situated in the CA-1 stratum orients were investigated, the larger basket cells close to stratum pyramidale and the smaller basket cells close to the alveus. Male Wistar rats were subjected to 20 min of transient cerebral ischemia by means of 4-vessel occlusion and perfusion fixed 1, 2, 4, or 21 days later. In both Golgi-impregnated and in routinely stained sections the pyramidal cells and interneurons in the hippocampal CA-1 region were examined and counted. The study clearly demonstrated the selective vulnerability of the CA-1 pyramidal cells, as no ischemic cell damage to or loss of interneurons was found.