Reversible Cerebral Ischemia Research Articles

Proteomic identification of an upregulated isoform of annexin A3 in the rat brain following reversible cerebral ischemia

We used proteomics to identify regulated proteins following cerebral ischemia in a rat model. Young rats were subjected to reversible middle cerebral artery (MCA) occlusion and proteins were extracted from the peri-infarcted and the corresponding contralateral area at days 3 and 14 postischemia. Proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. We report for the first time that an isoform of annexin A3 (ANXA3) was among the upregulated proteins in the postischemic rat brain. The results were confirmed by real-time PCR and by western blotting. Double- and triple-immunostaining with neuronal and microglia/macrophagic markers demonstrated that ANXA3 is produced by resting microglia in control tissue and by activated microglial/macrophage cells in the infarcted area. 3D-images of the infarcted area suggest that ANXA3 is associated with a phagocytic phenotype. Our study identifies ANXA3 as a novel marker of brain microglia, which should be of substantial value in future studies of microglial cells and its role in the postischemic brain.

Significance of Experimental Infarct Size as an Indicator of Therapeutic Efficacy in Humans

To the Editor: We would like to make a comment not in particular to the article by Shimamura et al1 but to the use of infarct size in animal experiments as a guide to human cerebrovascular research. Many human studies of treatments with neuroprotective agents for acute ischemic stroke, which had good experimental potential, had disappointing clinical results. This failure can be attributed to many factors.2 One of the main problems is that reliance on infarct size measurement alone in animals can be misleading as an indicator of therapeutic efficacy in humans.3 We know for example that nitric oxide synthase (NOS) inhibitors have provided significant neuroprotection in experimental animals subjected to focal ischemia.4,5 Tirilazad mesylate is a potent inhibitor of lipid peroxidation caused by …

A Reversible Middle Cerebral Artery Occlusion Model Using Intraluminal Balloon Technique in Monkeys

This study was designed to develop a simple, reversible primate middle cerebral artery (MCA) occlusion (MCAO) model. MCAO was achieved by inserting a specially designed intraluminal balloon catheter from the external carotid artery, then to the internal carotid artery, and finally into the proximal segment of MCA using 13 adolescent monkeys (Macaca mulatta) weighing 4.25 +/- 0.23 kg. The infarction was confirmed by magnetic resonance imaging at 3, 7, and 23 hours after MCAO. Local cerebral blood flow in the caudate nucleus decreased remarkably. Post-MCAO electroencephalographic monitoring showed the inhibition of electroencephalography with slow waves distinctly increasing. Neurologic evaluation demonstrated persistent deficits in the ischemic group. Neuropathologic examinations were consistent with the neurologic evaluations. The results suggested that this reversible monkey model resembled a human stroke and should offer a reasonable alternative to craniotomy and the extrinsic ligation of the MCA in the laboratory investigation of focal reversible cerebral ischemia.

Neuroprotective Effects of Virally Delivered HSPs in Experimental Stroke

Heat shock proteins (HSPs) are molecular chaperones with essential roles in modulating the proteolytic machinery and accelerating cell repair. Heat shock protein overexpression has been observed in vivo and in vitro under stresses including heat, nutrient deprivation and ischemia. Experiments in in vivo models of stroke indicate that transgenically overexpressed or virally delivered HSPs can enhance cell survival, but cannot always reduce lesion size. This study aims to assess the effects of virally delivered HSPs in a rat middle cerebral artery occlusion model of reversible focal cerebral ischemia using noninvasive magnetic resonance imaging. Attenuated herpes simplex virus carrying HSP27, HSP70, or a LacZ control was microinjected into the striatum 3 days before ischemia. Multislice T(2)-weighted images at 24 h after ischemia indicated that lesion volume was reduced by 44% in HSP27-treated animals compared with controls (P = 0.019). No significant differences were found between HSP70-treated and control animals (P = 0.88). Immunohistochemistry and Western blots revealed that HSP27 and HSP70 expression levels were equally high in injected hemispheres, but only the former had an effect on lesion size. This is the first evidence of the efficacy of gene therapy with any viral vector expressing HSP27 in an experimental model of stroke.

Gene therapy for stroke using viral delivery of heat schock proteins

Heat shock proteins (HSPs) are molecular chaperones with essential roles in cellular function such as modulation of the proteolytic machinery and acceleration of cell repair. This study uses MRI to assess the effects of pre-ischaemic viral delivery of HSPs on lesion size in a rat middle cerebral artery (MCA) occlusion model of reversible focal ischaemia. Perfusion maps of a 2-mm brain slice within the MCA territory were acquired to measure cerebral blood flow (CBF) and multislice T2-weighted scans were used to determine lesion volume 24 hours after ischaemia. Male rats were anaesthetised with 2% isoflurane in 100% O2. Viral suspensions (2.5 l of 2106 pfu) of herpes simplex virus carrying HSP27 (n=6), HSP70 (n=6), or LacZ (n=6) as a control, were stereotactically injected into rat striatum. Three days post-injection, rats were re-anaesthetised for middle cerebral artery occlusion (MCAO) by intraluminal insertion of a 290-m-suture past the MCA junction. After 30 minutes the suture was completely withdrawn to reperfuse the tissue and rats were allowed to recover for 24 hours before scanning. Coronal images were obtained approximately 0.5 mm from bregma on a 2.35 Tesla horizontal bore SMIS magnet with 4020 mm FOV, 2 mm slice thickness and 12864 pixels. T1 and CASL (continuous arterial spin labelling) EPI sequences were run for quantitative CBF mapping 3 and multislice T2-weighted SE images (1 mm slice thickness, 9 slices, TE=120 ms, TR=1500 ms) were acquired for lesion definition. All animals were imaged under general anaesthetic (halothane 2% in a 70:30 N2O:O2 mix) with physiological monitoring (ECG, rectal temperature). Three days later, the brains were extracted and immunohistochemistry and Western blots were carried out in order to verify expression levels of virally delivered HSPs in the brain. Total lesion volume was reduced by 44.8% (p=0.02) in HSP27 treated animals compared to controls whereas no significant differences were found between HSP70 treated and control animals (p=0.88). CASL maps indicated that there was no significant difference in relative CBF 24 hours after reperfusion in normal and ischaemic hemispheres between the groups that could account for differences in lesion size. Histological analysis of brain sections showed widespread staining for HSPs in basal ganglia and cortex. Western blots performed 72 hours after MCAO revealed that levels of HSP expression in HSP injected hemispheres were 4 times higher than in LacZ injected controls. In conclusion, we show that non–invasive MRI techniques can detect a significant reduction in lesion size after HSP27 gene delivery in a rat model of reversible focal cerebral ischaemia.

Hemodilution during cardiopulmonary bypass increases cerebral infarct volume after middle cerebral artery occlusion in rats.

Although the optimal hematocrit during cardiopulmonary bypass (CPB) is not defined, excessive hemodilution may lead to organ ischemia via a reduction in oxygen-carrying capacity uncompensated by autoregulatory and/or rheologic increases in organ blood flow. As a result, the consequences of hemodilution in patients at risk for cerebral ischemia are not clearly understood. We designed this study to evaluate the effects of hemodilution in the setting of focal cerebral ischemia during CPB. Wistar rats surgically prepared for CPB were randomized to either hemodilution (hemoglobin (Hb), 6 g/dL; n = 9) or control (Hb, 11 g/dL; n = 8) groups and subsequently exposed to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Immediately after the onset of MCAO (maintained for 90 min), 65 min of hypothermic (28 degrees C) CPB was initiated. Twenty-four hours later, functional neurological outcome and cerebral infarct volume were determined. Compared with controls, the hemodilution group had worse neurological performance (new score = 8 [2], hemodilution; versus 10 [2], control; P = 0.030) and larger total cerebral infarct volumes (182 +/- 84 mm(3), hemodilution; versus 103 +/- 58 mm(3), control; P = 0.043). In this experimental model of CPB with reversible MCAO-induced focal cerebral ischemia, hemodilution worsened neurological function and increased cerebral infarct volume.

Window of opportunity of cerebral hypothermia for postischemic white matter injury in the near-term fetal sheep.

Postresuscitation cerebral hypothermia is consistently neuroprotective in experimental preparations; however, its effects on white matter injury are poorly understood. Using a model of reversible cerebral ischemia in unanesthetized near-term fetal sheep, we examined the effects of cerebral hypothermia (fetal extradural temperature reduced from 39.4 +/- 0.1 degrees C to between 30 and 33 degrees C), induced at different times after reperfusion and continued for 72 hours after ischemia, on injury in the parasagittal white matter 5 days after ischemia. Cooling started within 90 minutes of reperfusion was associated with a significant increase in bioactive oligodendrocytes in the intragyral white matter compared with sham cooling (41 +/- 20 vs 18 +/- 11 per field, P < 0.05), increased myelin basic protein density and reduced expression of activated caspase-3 (14 +/- 12 vs 91 +/- 51, P < 0.05). Reactive microglia were profoundly suppressed compared with sham cooling (4 +/- 6 vs 38 +/- 18 per field, P < 0.05) with no effect on numbers of astrocytes. When cooling was delayed until 5.5 hours after reperfusion there was no significant effect on loss of oligodendrocytes (24 +/- 12 per field). In conclusion, hypothermia can effectively protect white matter after ischemia, but only if initiated early after the insult. Protection was closely associated with reduced expression of both activated caspase-3 and of reactive microglia.

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The role of p53-induced apoptosis in cerebral ischemia: effects of the p53 inhibitor pifithrin α

No neuroprotective compounds are clinically available for the treatment of ischemic stroke. The potential salutary effect of pifithrin α, a novel-specific inhibitor of the transcription factor p53, administered 1–6 h following focal reversible cerebral ischemia, was investigated. Studies measuring histological, motor, and behavioral outcomes showed significant improvements in pifithrin α-treated animals. Pifithrin α reduced the number of apoptotic cells in the ischemic brain by inhibiting the binding of p53 to its DNA sites as it reduced the expression of the p53-related gene p21 WAF without changing the amount of p53 protein itself.

Editorial Comment—Hyperglycemia and Early Reperfusion Therapy

Clinical studies of acute ischemic stroke support the conclusions of most experimental studies of focal cerebral ischemia in suggesting that admission hyperglycemia is associated with a worse clinical outcome.1 This association is more consistent in nonlacunar strokes2 and in experimental models of reversible focal cerebral ischemia.3 The acute ischemic penumbra might be preferentially susceptible to injury in hyperglycemic ischemia. Indeed, by use of MR methods, hyperglycemia in acute ischemic stroke was shown to promote the evolution of hypoperfused tissue to infarction and to do so by increased brain lactate …

HIF-1 alpha and VEGF expression after transient global cerebral ischemia.

Hypoxia inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) expression were studied in rat cerebral cortex after reversible global cerebral ischemia produced by cardiac arrest and resuscitation. Immunoblot analysis showed a significant induction of HIF-1 alpha protein after 1 hour of recovery from cardiac arrest which remained elevated for at least 12 hours. Upregulation of VEGF mRNA and protein were also observed but this was delayed in comparison to the HIF-1 alpha response. VEGF188 and VEGF164 mRNA levels were increased at 12-48 h of recovery from cardiac arrest but returned to basal expression after 7 days. Changes in VEGF120 mRNA expression did not reach statistical significance. Correspondingly, VEGF protein levels increased by about double at 24 and 48 hours of recovery but returned to basal levels after 7 days. These results suggest that cardiac arrest and resuscitation triggers HIF-1 alpha induction, which might be at least in part responsible for the stimulation of VEGF expression.

N‐acetyl cysteine protects against injury in animal model of transient focal cerebral ischemia

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long‐term disability worldwide (or developing countries). The purpose of this study is to investigate the therapeutic potential of N‐acetyl cysteine (NAC), a precursor of glutathione, and a potent antioxidant, in tissue protection against transient focal cerebral ischemia model in rats. Our laboratory has already demonstrated the efficacy of NAC against induction of proinflammatory cytokines, iNOS, and production of NO in cultured cells. In this study, 250–300 g male Sprague–Dawley rats were subjected to 45 min of reversible focal cerebral ischemia through middle cerebral artery (MCA) occlusion. The protective effect of NAC is supported by reduced infarct volume as well as the suppression of TNF‐α and iNOS in the treated animals. Pretreatment with NAC reduced the infarct size by 49.6% as compared to the saline treated group along with reducing the neurological evaluation score by more than 50%. Our results suggest that preadministration of NAC attenuates focal cerebral ischemia by protecting cells against free radical damage.Acknowledgements: Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.

Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity.

Heparin and heparinoids have long been proposed for stroke treatment. This study investigates the effect of enoxaparin (Lovenox, Clexane), a low-molecular-weight heparin, on functional outcome (neuroscore) and lesion size in stroke models with reversible and irreversible cerebral ischemia using middle cerebral artery occlusion (MCAO) in the rat. Ischemia was induced in rats by transient occlusion for 2 hours or by permanent electrocoagulation of the left MCA. Forty-eight hours after ischemia, neurological deficit was evaluated by scoring sensorimotor functions and ischemic damage was quantified by histological evaluation of lesion volumes. After transient MCAO, enoxaparin at 2x1.5 mg/kg IV (2 and 24 hours after insult) significantly reduced lesion size by 30% (P<0.05) and improved neuroscore (P<0.01). This significant effect on lesion size and neuroscore was still evident when treatment was started 5 hours after insult. Administered under the same protocol with a 5 hours delay post permanent MCAO, enoxaparin reduced lesion size by 49% (P<0.05) and improved neuroscore (P<0.01). This study indicates that standard nonhemorrhagic doses of enoxaparin reduce ischemic damage with a wide therapeutic window. In addition to its anticoagulant properties, other properties of enoxaparin could act in synergy to explain its neuroprotective profile in ischemia. Thus clinical application of enoxaparin treatment in stroke warrants serious consideration.

Dose-dependent neuroprotection with tiagabine in a focal cerebral ischemia model in rat.

Enhancement of the inhibitory activity of GABA may protect ischemic neurons. We evaluated the neuroprotective effect of tiagabine, a novel GABA agonist, in reversible focal cerebral ischemia rats subjected to 2h middle cerebral artery (MCA) occlusion. Tiagabine was given at 10, 20 and 40 mg/kg, i.p., 2 h after the onset of reperfusion. We found that post-ischemia treatment with tiagabine improved neurobehavioral outcome and reduced brain infarction volume in a dose-dependent manner. The data suggest that post-ischemic administration of tiagabine is neuroprotective in the focal cerebral ischemia model.

Attenuation of free radical generation during reversible focal cerebral ischemia with the nitric oxide inhibitor, l-NAME ( l- NG-nitro- l-arginine methyl ester)

The role of oxygen free radical generation during reversible focal cerebral ischemia and its relationship to nitric oxide mediated mechanisms were examined. In this study, a left frontal cortex microdialysis probe was placed into the previously defined ischemic penumbra region and perfused with a salicylate/CSF solution in the presence or absence of the nitric oxide synthase (NOS) inhibitor l-NAME. Rats were then subjected to transient left hemisphere focal cerebral ischemia. Dialysate was collected at baseline and during the ischemic/reperfusion phase, and the hydroxylation products of salicylate were measured by HPLC with electrochemical detection. A significant elevation of free radical adduct formation was observed in the penumbra region during ischemia/reperfusion. This elevation was significantly attenuated by l-NAME during the reperfusion phase. Elevation of free radical adduct formation within the penumbra region during cerebral ischemia/reperfusion may be mediated in part by NOS-dependent mechanisms.

Vascular endothelial growth factor upregulation in transient global ischemia induced by cardiac arrest and resuscitation in rat brain

This study examined vascular endothelial growth factor (VEGF) expression in rat brain after reversible global cerebral ischemia produced by cardiac arrest and resuscitation. Three alternative splicing forms, VEGF 188, VEGF 164 and VEGF 120, were observed in cortex, hippocampus and brainstem by RT–PCR analysis. After 24 h of recovery from cardiac arrest, mRNA levels corresponding to VEGF 188 and VEGF 164 were significantly increased by about double in all the regions analyzed. These mRNA levels remained elevated at 24 and 48 h of recovery but returned to basal expression after 7 days of recovery. Changes in VEGF 120 expression after cardiac arrest did not reach statistical significance. VEGF protein expression measured by Western blot was also increased by about double at 24 and 48 h of recovery but returned to control levels after 7 days of recovery. VEGF immunohistochemistry localized this increased expression mostly associated with astrocytes. Considering its biological activity, VEGF induction after cardiac arrest and resuscitation may be responsible for the increased vascular permeability and the resultant vasogenic edema, found 24–48 h after reversible global ischemia.

Quantitative T*2 and T?2 maps during reversible focal cerebral ischemia in rats: Separation of blood oxygenation from nonsusceptibility-based contributions

Quantitative imaging contrast is evaluated which allows the selective measurement of the blood oxygenation state during cerebral ischemia within a multiparametric imaging study on rats. In a first step, the ambiguities arising in T*(2)-weighted images due to T*(2) heterogeneity are eliminated by calculating T*(2) maps. Then, 1/T'(2) maps are calculated according to 1/T'(2) = 1/T*(2) - 1/T(2) to eliminate nonsusceptibility-induced changes of 1/T*(2) after the induction of stroke. This is of particular importance in the presence of vasogenic edema. The changes Delta(1/T'(2)) after the onset of ischemia selectively quantify the variations of the deoxyhemoglobin content during the development of the infarct. The presented results are not available from conventionally recorded parameters of a stroke study and, together with perfusion-weighted images, form a powerful combination to analyze the oxygen consumption and the metabolism of the tissue. Magn Reson Med 42:1027-1032, 1999.

A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury.

Various studies describe increased concentrations of transforming growth factor-beta (TGF-beta) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-beta after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-beta produced after an episode of cerebral ischemia by injecting a soluble TGF-beta type II receptor fused with the Fc region of a human immunoglobulin (TbetaRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-beta. Then, the authors tested its ability to reverse the effect of TGF-beta1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 microg/mL of TbetaRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-beta1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-beta1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-beta1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 microg of TbetaRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-beta1 mRNA. Furthermore, when added to the excitotoxin, TbetaRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TbetaRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-beta1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 +/- 9.5 versus 152.8 +/- 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-beta in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-beta1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

Quantitative T*(2) and T'(2) maps during reversible focal cerebral ischemia in rats: separation of blood oxygenation from nonsusceptibility-based contributions.

Quantitative imaging contrast is evaluated which allows the selective measurement of the blood oxygenation state during cerebral ischemia within a multiparametric imaging study on rats. In a first step, the ambiguities arising in T*(2)-weighted images due to T*(2) heterogeneity are eliminated by calculating T*(2) maps. Then, 1/T'(2) maps are calculated according to 1/T'(2) = 1/T*(2) - 1/T(2) to eliminate nonsusceptibility-induced changes of 1/T*(2) after the induction of stroke. This is of particular importance in the presence of vasogenic edema. The changes Delta(1/T'(2)) after the onset of ischemia selectively quantify the variations of the deoxyhemoglobin content during the development of the infarct. The presented results are not available from conventionally recorded parameters of a stroke study and, together with perfusion-weighted images, form a powerful combination to analyze the oxygen consumption and the metabolism of the tissue. Magn Reson Med 42:1027-1032, 1999.

Increased intracellular calpain detection in experimental focal cerebral ischemia.

Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.