Minutes Of Global Cerebral Ischemia Research Articles

Lung-Derived SOD3 Attenuates Neurovascular Injury After Transient Global Cerebral Ischemia.

BackgroundSystemic innate immune priming is a recognized sequela of post‐ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia‐reperfusion.Methods and ResultsFollowing 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild‐type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood‐brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood‐brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation.ConclusionsIn addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia‐reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung‐neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.

Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia.

Decreased blood flow in the brain leads to a rapid increase in reactive oxygen species (ROS). NADPH oxidase (NOX) is an enzyme family that has the physiological function to produce ROS. NOX2 and NOX4 overexpression is associated with aggravated ischemic injury, while NOX2/4-deficient mice had reduced stroke size. Dysregulation of matrix metalloproteinases (MMPs) contributes to tissue damage. The active form of vitamin D3 expresses neuroprotective, immunomodulatory, and anti-inflammatory effects in the CNS. The present study examines the effects of the vitamin D3 pretreatment on the oxidative stress parameters and the expression of NOX subunits, MMP9, microglial marker Iba1, and vitamin D receptor (VDR), in the cortex and hippocampus of Mongolian gerbils subjected to ten minutes of global cerebral ischemia, followed by 24 hours of reperfusion. The ischemia/reperfusion procedure has induced oxidative stress, changes in the expression of NOX2 subunits and MMP9 in the brain, and increased MMP9 activity in the serum of experimental animals. Pretreatment with vitamin D3 was especially effective on NOX2 subunits, MMP9, and the level of malondialdehyde and superoxide anion. These results outline the significance of the NOX and MMP9 investigation in brain ischemia and the importance of adequate vitamin D supplementation in ameliorating the injury caused by I/R.

Abstract WMP120: Long-Term Estrogen Deprivation Enhances Hippocampal Amyloidogenic Processing and Cognitive Decline after Global Cerebral Ischemia

Women who enter menopause prematurely have a doubled lifetime risk of cognitive decline and dementia. The mechanism underlying this phenomenon is unclear, but it is postulated to be associated with dramatic and prolonged deprivation of the neuroprotective ovarian hormone 17beta-estradiol (E2). To shed light on this issue, 3-month-old female Sprague Dawley rats were bilaterally ovariectomized and had subcutaneous, osmotic mini-pumps containing a low, Diestrus I dose of E2 implanted either immediately (short-term E2 deprivation - STED) or 10 weeks later (long-term E2 deprivation - LTED). One week following continuous E2 replacement, all animals were subjected to 10 minutes of global cerebral ischemia (GCI) and sacrificed for tissue harvest. Intriguingly, we observed a profound induction of Alzheimer’s disease-related proteins (beta-amyloid and hyperphosphorylated-tau) throughout the hippocampus of LTED females who experienced GCI. In the same animals, we also noted a switch to amyloidogenic processing of amyloid precursor protein (APP), such that hippocampal expression of beta-amyloid cleaving enzyme 1 (BACE1) was enhanced and hippocampal expression of A D isintegrin A nd M etalloprotease 17 (ADAM17) was diminished. Hippocampal expression of an alternate alpha secretase, ADAM10, remained unchanged. Furthermore, LTED females demonstrated enhanced post-ischemic cognitive decline, as they performed worse than STED females on the Morris Water Maze after GCI. Importantly, while immediate replacement of ovarian E2 was capable of preventing these events in STED females, delayed treatment with low-dose E2 was ineffective. Finally, c-Jun N-terminal Kinase (JNK) was identified as a critical mediator of post-ischemic amyloidogenesis, cognitive decline, and apoptosis in LTED females, as delayed treatment with a JNK inhibitor was able to prevent these events. These data could help explain why surgically menopausal women have an increased risk of cognitive decline and dementia and why postmenopausal women fare worse than age-matched men following a stroke. Furthermore, they lend support to the critical period hypothesis, suggesting that only perimenopausal replacement of ovarian E2 will provide neurological benefit for post-menopausal women.

Kynurenic acid attenuates NMDA-induced pial arteriolar dilation in newborn pigs

The excitatory amino acid glutamate is a potent vasodilator in the central nervous system. Glutamate-induced vasodilation is mediated primarily by N-methyl- d-aspartate (NMDA) and AMPA/kainate (KAIN) receptors. We have now tested whether two metabolites of the kynurenine pathway of tryptophan degradation acting at the NMDA receptor, the antagonist kynurenic acid (KYNA) and the agonist quinolinic acid (QUIN), are capable of modulating the dilation of pial arterioles. The closed cranial window technique was used, and changes in vessel diameter (∼100 μm) were analyzed in anesthetized newborn piglets. Topical application of NMDA (10 −4 M) or KAIN (5 × 10 −5 M) resulted in marked vasodilation (44 ± 5% and 39 ± 4%, respectively). Neither KYNA nor QUIN (both at 10 −5 to 10 −3 M) affected the vessel diameter when applied alone. Co-application of KYNA dose-dependently reduced the vasodilation caused by 10 −4 M NMDA and also attenuated the KAIN-induced response. Ten minutes of global cerebral ischemia did not modify the interaction between KAIN and KYNA. In contrast, KYNA did not affect vasodilation to hypercapnia, elicited by the inhalation of 10% CO 2. Moreover, endogenous levels of KYNA and QUIN in the cerebral cortex, hippocampus and thalamus were found to be essentially unchanged during the early reperfusion period (0.5–2 h) following an episode of cerebral ischemia. Our data are relevant for the use of drugs that target the kynurenine pathway for therapeutic interventions in cerebrovascular diseases.

The Effects of Emopamil on Neurologic and Neuropathologic Outcomes Following Transient Global Ischemia in Rabbits

Background: Previous studies have suggested that the phenylalkylamine calcium channel blocker, emopamil, may have neuroprotective properties. This study was designed to examine the effects of emopamil on the neurologic and neuropathologic outcome after transient global cerebral ischemia. Methods: New Zealand white rabbits were randomly assigned to one of the following groups: emopamil group (n = 6; emopamil 1 mg/kg bolus prior to ischemia and then 0.1 mg/kg/min for 1 hour), hypothermia group (n = 7; 29oC prior to, during and until 1 hour after ischemia), or control group (n = 7). All rabbits were subjected to 6.25 minutes of global cerebral ischemia produced by a neck tourniquet inflation (20 psi) combined with hypotension during halothane anesthesia. Neurologic deficit scoring was done at 24 hours and 48 hours after ischemia. The rabbits were then euthanized and perfused with 10% formalin. Coronal sections of brain (6m) were stained with hematoxylin and eosin. The sections were scored for evidence of ischemic injury by two observers blinded as to the experimental group. Results: The hypothermia group had significantly less evidence of neurologic and neuropathologic injury when compared to the control group (P 0.05). There were no significant differences between the control and emopamil groups in neurologic or neuropathologic outcome. Conclusions: These results suggest that the peri-ischemic administration of emopamil had no effect on their neurologic or neuropathologic outcome in this model, but, as anticipated, hypothermia was neuroprotective.

Microarray analysis of hippocampal gene expression in global cerebral ischemia.

The brain's response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotide-based microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7-fold or greater increases in the expression of 57 genes and 1.7-fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injury-response genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1; the IGF1 receptor and the IGF1-binding protein IGFBP2; Rb, the Rb-binding protein E2F1, and the E2F-related transcription factor, TFDP1; the CACNB3 and CACNB4 beta-subunits of the voltage-gated calcium channel; and caspase-3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemia-GRB2, an adapter protein involved in growth-factor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain.

Histopathologic consequences of hyperglycemic cerebral ischemia during hypothermic cardiopulmonary bypass in pigs

Background. This study examined whether 34°C or 31°C hypothermia during global cerebral ischemia with hyperglycemic cardiopulmonary bypass (CPB) in surviving pigs improves electroencephalographic (EEG) recovery and histopathologic scores when compared with normothermic animals. Methods. Anesthetized pigs were placed on CPB and randomly assigned to 37°C (n = 9), 34°C (n = 10), or 31°C (n = 8) management. After increasing serum glucose to 300 mg/dL, animals underwent 15 minutes of global cerebral ischemia by temporarily occluding the innominate and left subclavian arteries. Following reperfusion, rewarming, and termination of CPB, animals were recovered for 24 (37°C animals) or 72 hours (34°C and 31°C animals). Daily EEG signals were recorded, and brain histopathology from cortical, hippocampal, and cerebellar regions was graded by an independent observer. Results. Before ischemia, serum glucose concentrations were similar in the 37°C (307 ± 9 mg/dL), 34°C (311 ± 14 mg/dL), and 31°C (310 ± 15) groups. By the first postoperative day, EEG scores in 31°C animals (4.2 ± 0.6) had returned to baseline and were greater than those in the 34°C (3.4 ± 0.5) and 37°C (2.5 ± 0.4) groups ( p < 0.05, respectively, between groups). Cooling to 34°C showed selective improvement over 37°C in hippocampal, temporal cortical, and cerebellar regions, but the greatest improvement in all regions occurred with 31°C. Cumulative neuropathology scores in 31°C animals (13.5 ± 2.2) exceeded 34°C (6.8 ± 2.2) and 37°C (1.9 ± 2.1) animals ( p < 0.05, respectively, between groups). Conclusions. Hypothermia during CPB significantly reduced the morphologic consequences of severe, temporary cerebral ischemia under hyperglycemic conditions, with the greatest protection at 31°C.

Tolerance-Inducing dose of 3-nitropropionic acid modulates bcl-2 and bax balance in the rat brain: a potential mechanism of chemical preconditioning.

Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax. two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia-reperfusion. and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia-reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; "chemical preconditioning") or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro- and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro- and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical preconditioning using 3-NPA in rats.

Laser Doppler scanning: how many measurements are required to assess regional cerebral blood flow?

This study was initiated to determine the optimal number of measuring sites necessary to estimate regional cerebral blood flow (CBF) under pathophysiological conditions. 25 rats were exposed to 15 minutes of global cerebral ischemia. Local CBF was sequentially measured by laser Doppler (LD) at 32 locations during baseline conditions, ischemia and reperfusion using a computer-controlled scanning technique. A simulation study was performed based on 800 local measurements at each time point: random samples (size 1-100) were repeatedly drawn to estimate the variability of median flow. Accuracy was defined as probability that the simulated median differed less than +/- 5 LD-units from the true median of the 800 data. Above a single location, CBF was measured with an accuracy of 21.6 +/- 0.4% (baseline conditions, n = 100 simulations, mean +/- SEM), 85.8 +/- 0.4% (ischemia) and 11.1 +/- 0.3% (30th min. reperfusion). Accuracy increases to 75.2 +/- 0.5% (baseline conditions), 100 +/- 0% (ischemia) and 41.8 +/- 0.6% (30th min. reperfusion) if 24 locations are scanned. Scanning, therefore, improves accuracy and reduces variability of CBF measurements. With enough local CBF measurements laser Doppler assessment of regional CBF is possible. Single location CBF assessment is sufficiently accurate only during ischemia. During reperfusion, when accuracy is half reduced compared to baseline conditions, larger sample sizes are required.

Mitochondrial potassium channel opener diazoxide preserves neuronal-vascular function after cerebral ischemia in newborn pigs.

N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K(ATP)) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K(ATP) channels (mitoK(ATP)) are involved in the ischemic preconditioning-like effect of K(+) channel openers. We determined whether the selective mitoK(ATP) channel opener diazoxide preserves the vascular dilation to NMDA after I/R. Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 micromol/L diazoxide; and coapplication of 100 micromol/L 5-hydroxydecanoic acid (5-HD), a K(ATP) antagonist with diazoxide. NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 micromol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 micromol/L NMDA-induced vasodilation after I/R was 53+/-19% (mean+/-SEM, n=8) in vehicle-treated controls versus 55+/-10%, 85+/-5%, and 99+/-15% in animals pretreated with 1, 5, and 10 micromol/L diazoxide (n=8, n=8, and n=12, respectively) and 60+/-15% in the group treated with 5-HD+diazoxide (n=5). The mitoK(ATP) channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK(ATP) channels may play a role in mediating the protective effect of other K(+) channel openers.

Breakdown of Calcium Homeostasis in Relation to Tissue Depolarization: Comparison between Gray and White Matter Ischemia

In vitro studies suggest that ischemic injury of cerebral white matter is mediated by nonsynaptic cellular mechanisms, such as Ca2+ entry into axons through reversal of the Na+ -Ca2+ exchanger. The authors investigated extracellular Ca2+ concentration in relation to tissue depolarization (direct current potential) in vivo using ion-selective electrodes in cortical gray and subcortical white matter of alpha-chloralose-anesthetized cats during 120 minutes of global cerebral ischemia. On induction of ischemia, regional CBF, as measured by hydrogen clearance, ceased. The direct current potential decreased rapidly within minutes in gray matter and with little time delay in white matter. Extracellular Ca2+ concentration decreased just as quickly in gray matter. In white matter, in contrast, extracellular Ca2+ increased in the first 20 to 30 minutes, and a delayed and much slower decline, compared with gray matter, was observed thereafter, reaching a minimal level only about 60 minutes after occlusion. Our results suggest that smaller and delayed transmembrane shifts of Ca2+ are correlates of delayed ischemic membrane dysfunction in central white matter tracts, which may be explained by a lack of synaptic mechanisms.

Lamotrigine attenuates cortical glutamate release during global cerebral ischemia in pigs on cardiopulmonary bypass.

The dose-response effects of pretreatment with lamotrigine (a phenyltriazine derivative that inhibits neuronal glutamate release) in a porcine cerebral ischemia model during cardiopulmonary bypass were studied. Sagittal sinus catheters and cortical microdialysis catheters were inserted into anesthetized pigs. Animals undergoing normothermic cardiopulmonary bypass were pretreated with lamotrigine 0, 10, 25, or 50 mg/kg (n = 10 per group). Fifteen minutes of global cerebral ischemia was produced, followed by 40 min of reperfusion and discontinuation of cardiopulmonary bypass. Cerebral oxygen metabolism was calculated using cerebral blood flow (radioactive microspheres) and arterial-venous oxygen content gradients. Concentrations of microdialysate glutamate and aspartate were quantified; electroencephalographic signals were recorded. After cardiopulmonary bypass, blood and cerebrospinal fluid were sampled for S-100B protein, and a biopsy was performed on the cerebral cortex for metabolic profile. Lamotrigine caused dose-dependent reductions in systemic vascular resistance so that additional fluid was required to maintain venous return. Concentrations of glutamate and aspartate did not change during reperfusion after 50 mg/kg lamotrigine in contrast to fivefold and twofold increases, respectively, with lower doses. There were no intergroup differences in cerebral metabolism, electroencephalographic scores, cortical metabolites, brain lactate, or S-100B protein concentrations in the cerebrospinal fluid and blood. Lamotrigine 50 mg/kg significantly attenuated excitatory neurotransmitter release during normothermic cerebral ischemia during cardiopulmonary bypass without improving other neurologic parameters. Lamotrigine caused arterial and venous dilation, which limits its clinical usefulness.

Density of serotonin receptors in rat brain following global cerebral ischemia: An autoradiographic study

Serotonin-1A receptor antagonists and serotonin-2A receptor agonists have in several studies been shown to have neuroprotective properties in roden models of cerebral ischemia A quantitative autoradiographic study was made to determine the density of serotonin-1A and serotonin-2A receptors in rat brain subjected to 10 minutes of global cerebral ischemia. The ischemic group and control group was sacrificed 24 hours after the ischemic attack. In the ischemic group of rats the density of serotonin-1A receptors was reduced in ventral dentate gyrus, lateral reticular thalamic nucleus and cortex compared to the control group. The changes in the lateral reticular thalamic nucleus and in cortex can be explained as a reflection of cell loss in these areas. However, the reduction in ventral dentate gyrus is unrelated to cell loss detected by eosin-hematoxylin staining. No changes between the to groups was seen for the serotonin-2A receptor.

Density of serotonin receptors in rat brain following global cerebral ischemia: An autoradiographic study

Serotonin-1A receptor antagonists and serotonin-2A receptor agonists have in several studies been shown to have neuroprotective properties in roden models of cerebral ischemia A quantitative autoradiographic study was made to determine the density of serotonin-1A and serotonin-2A receptors in rat brain subjected to 10 minutes of global cerebral ischemia. The ischemic group and control group was sacrificed 24 hours after the ischemic attack. In the ischemic group of rats the density of serotonin-1A receptors was reduced in ventral dentate gyrus, lateral reticular thalamic nucleus and cortex compared to the control group. The changes in the lateral reticular thalamic nucleus and in cortex can be explained as a reflection of cell loss in these areas. However, the reduction in ventral dentate gyrus is unrelated to cell loss detected by eosin-hematoxylin staining. No changes between the to groups was seen for the serotonin-2A receptor.

Postischemic application of lipid peroxidation inhibitor U-101033E reduces neuronal damage after global cerebral ischemia in rats.

The lipid peroxidation inhibitor U-101033E was examined for effects on cerebral blood flow (CBF), cortical tissue hemoglobin oxygen saturation (HbSo2), and neuronal damage. Fifteen minutes of global cerebral ischemia was induced by two-vessel occlusion and hypobaric hypotension. Wistar rats (n = 25) were randomized to receive vehicle (n = 9) or 40 mg/kg U-101033E (n = 9) intraperitoneally during 2 hours of reperfusion. A sham group (n = 7) had neither ischemia nor therapy. Histology was evaluated 7 days after ischemia. During late hyperperfusion (at 17 minutes), vehicle-treated animals had a higher (P = 0.044) cortical tissue HbSo2 (72.0 +/- 1.4%) than did U-101033E-treated animals (65.8 +/- 2.5%). Neuronal counts in the superficial cortex layer found after 7 days correlated negatively with rCBF (r = -0.76; P < 0.001) or cortical tissue HbSo2 (r = -0.56; P = 0.028) assessed during the late hyperperfusion phase. U-101033E reduced neuronal damage in hippocampal CA1 from 64.3 +/- 9.2% to 31.2 +/- 8.4% (P = 0.020), as well as in the superficial cortical layer from 53.5 +/- 14.6% to 12.8 +/- 11.7% (P = 0.046). While animals in the vehicle group had reduced counts in all four examined cortex layers (P < 0.05 versus sham group), there was significant cortical neuron loss in the U-101033E group in only one of four areas. U-101033E had no effect on resting CBF or CO2 reactivity. Postischemic application of U-101033E protects hippocampal CA1 and cortical neurons after 15 minutes of global cerebral ischemia. The results indicate that free radical-induced lipid peroxidation contributes to reperfusion injury, a process that can be inhibited by antioxidants such as U-101033E.

Correlation Between CSF and Serum S-100 Protein after Cerebral Ischemia in Pigs on Cardiopulmonary Bypass

Introduction: One potential marker of central nervous system damage during cardiac surgery is the astroglial protein S-100, which has been shown to increase with the duration of cardiopulmonary bypass (CPB) [1]. However, recovery of the S-100 protein in blood has not been directly correlated with functional or histopathological outcomes after CPB. Part of the diagnostic problem may be related to whether concentration of this protein marker in the serum correlates with cerebrospinal fluid (CSF). In a porcine model of global cerebral ischemia during CPB, we examined the presence of S-100 in the sagittal sinus after bypass and correlated serum and CSF S-100 levels. Methods: In an approved study, sagittal sinus catheters were placed in 40 anesthetized pigs. Animals were cannulated and placed on CPB. Baseline serum S-100 levels from the sagittal sinus were obtained. Electroencephalographic (EEG) signals from scalp needle electrodes assessed the severity of cerebral ischemia. All animals underwent 15 minutes of global cerebral ischemia produced by temporarily ligating the innominate and left subclavian arteries followed by reperfusion. Thirty minutes after discontinuing CPB, a needle was inserted into the cisterna magna to collect CSF; simultaneously, a sample of sagittal sinus blood was aspirated. S-100 protein contents were analyzed using a monoclonal immunoradiometric assay (Sangtec Medical, Sweden). Results: Cerebral ischemia was confirmed by total loss of EEG activity. Sagittal sinus S-100 levels at baseline were 0.09 +/- 0.06 [micro sign]g/L and increased significantly after ischemia (0.24 +/- 0.22 [micro sign]g/L; p<0.05). After bypass, CSF S100 levels were significantly greater than corresponding serum concentrations: 4.35 +/- 3.1 [micro sign]g/L (p<0.05). There was a weak linear correlation (r=0.37) between serum and CSF S-100 concentrations after CPB (p<0.02; Figure 1).Figure 1Discussion: Serum S-100 protein in the sagittal sinus increased significantly after ischemia, confirming the usefulness this assay to detect cerebral ischemic injury during CPB. However, the variable relationship between blood and CSF sites may indicate short-lived, reversible changes in blood-brain barrier permeability after ischemia on CPB. The amount of ischemic cerebral damage as measured by CSF S-100 protein concentration may not be reflected by serum S-100 values.

Hyperbaric oxygen after global cerebral ischemia in rabbits reduces brain vascular permeability and blood flow.

Hyperbaric oxygen (HBO) has been advocated as a therapy to improve neurological recovery after ischemia, since HBO may improve tissue oxygen delivery. We examined the effect of HBO treatment after global cerebral ischemia on early brain injury. Rabbits were subjected to 10 minutes of global cerebral ischemia by cerebrospinal fluid compression. After 30 minutes of reperfusion, rabbits either were subjected to HBO for 125 minutes and then breathed 100% O2 at ambient pressure for 90 minutes or breathed 100% O2 for 215 minutes. At the end of reperfusion and 90 minutes after exposure, brain vascular permeability and cerebral blood flow were measured. Somatosensory evoked potentials were monitored throughout the experiment. HBO treatment reduced (P < .05) brain vascular permeability by 16% in gray matter and by 20% in white matter. Cerebral blood flow was lower (P < .05) in the HBO group (40.9 +/- 1.9 mL/min per 100 g, mean +/- SEM) compared with controls (50.8 +/- 2.0 mL/min per 100 g). Somatosensory evoked potential recovery was similar in the two groups (P > .05). HBO administered after global cerebral ischemia promoted blood-brain barrier integrity. HBO treatment also reduced cerebral blood flow; this effect was not associated with a reduction in evoked potential recovery. Since neurological outcome after global cerebral ischemia is generally poor and treatment options are limited, HBO should be further investigated as a potential therapy.

Transient brain ischemia in rabbits: the effect of ω-conopeptide MVIIC on hippocampal excitatory amino acids

Neurologic injury that occurs after ischemia results from a cascade of events involving the release of various endogenous neurotoxins. A portion of the release of excitatory neurotransmitters is calcium dependent and may be attenuated by administration of calcium channel blockers. Using an in vivo model of ischemia, we studied the effects of ω-conopeptide MVIIC, a voltage-sensitive calcium channel blocker, and hypothermia (32°C) on hippocampal glutamate and aspartate release in the peri-ischemic period. Thirty-four New Zealand white rabbits of either sex were anesthetized with halothane, intubated, and mechanically ventilated. Monitored variables included blood gases, mean arterial blood pressure, and the electroencephalogram. Microdialysis catheters were transversely inserted through the anterior portion of the dorsal hippocampus and perfused with artificial cerebrospinal fluid at a rate of 2 μl/min. After stabilization period, animals were randomly assigned to one of the following groups: Control group ( n = 8), 10 μM ω-conopeptide MVIIC group ( n = 7), 100 μM ω-conopeptide MVIIC group ( n = 7), Hypothermia group ( n = 6; cranial temperature = 32°C), and ω-conopeptide MVIIC + hypothermia group ( n = 6; 100 μM ω-conopeptide MVIIC and cranial temperature 32°C). All the rabbits were subjected to 10 minutes of global cerebral ischemia produced by neck tourniquet inflation combined with hypotension during halothane anesthesia. Conopeptide MVIIC was administered in the artificial cerebrospinal fluid used to perfuse the microdialysis catheter. In control animals, ischemia caused a significant increase in glutamate (9.7 fold) and aspartate (11.3 fold) concentrations. This increase was markedly attenuated ( P < 0.05) in all treatment groups (MVIIC 10 μM, 100 μM, hypothemia, and MVIIC + hypothermia). These results demonstrate that ω-conopeptide MVIIC (10 μM and 100 μM) can attenuate ischemia-induced increases of glutamate and aspartate concentrations in the peri-ischemic period. This effect is probably caused by a blockade of presynaptic calcium channels and decreased synaptosomal release of excitatory neurotransmitters.

Brain Temperature Modulations During Global Ischemia Fail to Influence Extracellular Lactate Levels in Rats

While brain tissue lactate increases during cerebral ischemia and is known to be important in the pathogenesis of ischemic brain injury, patterns of extracellular lactate accumulation have been less well characterized, and the influence of brain temperature has not been previously investigated. Mild brain temperature modulations are known to affect the outcome of ischemia dramatically. This study examined changes of extracellular lactate during and after global cerebral ischemia, in which intraischemic brain temperature was held at either 30 degrees C, 37 degrees C, or 39 degrees C. Halothane-anesthetized fasted male Wistar rats underwent 20 minutes of global cerebral ischemia produced by bilateral carotid artery occlusions plus systemic hypotension (40 to 50 mm Hg). Rectal temperature was maintained at 37 degrees C throughout, and intraischemic brain temperature was held at either 30 degrees C (n = 6), 37 degrees C (n = 5), or 39 degrees C (n = 5). Before and after the ischemic insult, brain temperature was maintained at 37 degrees C in all groups. A microdialysis cannula was implanted in the right dorsolateral striatum and perfused with Ringer's solution. Dialysate samples were collected at 10-minute intervals before, during, and after ischemia and were analyzed for lactate by enzymatic-fluorometric techniques. In all groups, extracellular lactate rose during ischemia and peaked at 10 to 30 minutes of recirculation. Maximal extracellular lactate elevations were sevenfold, eightfold, and eightfold above control in the 30 degrees C, 37 degrees C, 39 degrees C groups, respectively. Significant elevations with respect to control were observed in all groups at 10 to 30 minutes of recirculation. In the 30 degrees C group, these elevations above control were also significant at the 10- and 20-minute ischemic time points (P = .001). At 30 minutes of recirculation, however, lactate levels were lower in the 30 degrees C rats than in the other groups. These data provide evidence that extracellular lactate accumulation is not a crucial determinant of ischemic brain injury. Our results suggest that the increased lactate release during ischemia and the accelerated clearance of lactate during recirculation might contribute in part to the neuroprotection of intraischemic hypothermia.

Neuroprotective Properties of the Novel Antiepileptic Lamotrigine in a Gerbil Model of Global Cerebral Ischemia

Elevated glutamate levels are thought to be a primary cause of neuronal death after global cerebral ischemia. The purpose of this study was to investigate the potential neuroprotective effects of lamotrigine, a novel antiepileptic drug that inhibits the release of glutamate in vitro, with both behavioral and histological measures of global ischemia in gerbils. The common carotid arteries of gerbils were occluded for either 5, 10, or 15 minutes. Twenty-one days after reperfusion, gerbils were tested for impairments in a spatial memory task (Morris water maze). After water maze testing the animals were killed, and damage to hippocampal pyramidal cells was assessed. The effect of lamotrigine on the behavioral and histological outcome of either 5 or 15 minutes of global ischemia was evaluated. Bilateral occlusion of the common carotid arteries for 5 minutes resulted in severe degeneration of hippocampal CA1 and CA2 pyramidal cells. Lamotrigine significantly prevented loss of hippocampal CA1 neurons when administered acutely (100 mg/kg PO) immediately after reperfusion or when administered in two equal doses of 30 or 50 mg/kg 2 hours before and immediately after reperfusion. Gerbils subjected to 5 minutes of ischemic insult were not impaired in their ability to solve a spatial memory task 21 days after cerebral ischemia. However, gerbils subjected to 10 and 15 minutes of carotid artery occlusion showed significant impairment in their ability to solve a water maze task. Lamotrigine significantly protected against the cognitive deficits associated with 15 minutes of cerebral ischemia. Histologically, increased durations of cerebral ischemia resulted in a progressive loss of CA1, CA2, and CA3 pyramidal cells. Lamotrigine completely protected gerbils exposed to 15 minutes of cerebral ischemia against CA3 cell loss and greatly reduced damage to the CA1 and CA2 cell tracts of the hippocampus. Lamotrigine also reduced the mortality associated with 15 minutes of ischemia. Lamotrigine had neuroprotective effects in a gerbil model of global cerebral ischemia. Lamotrigine protected gerbils against behavioral deficits resulting from 15 minutes of carotid occlusion and also prevented histological damage resulting from 5 and 15 minutes of global cerebral ischemia.