Experimental Middle Cerebral Artery Occlusion Research Articles

Extensive complex neocortical movement topography devolves to simple output following experimental stroke in mice.

The neocortex encodes complex and simple motor outputs in all mammalian species that have been tested. Given that changes in neocortical reorganization (and corresponding corticospinal output) have been implicated in long term motor recovery after stroke injury, there remains a need to understand this biology in order to expedite and optimize clinical care. Here, changes in the neocortical topography of complex and simple movement outputs were evaluated in mice following experimental middle cerebral artery occlusion (MCAo). Neocortical motor output was defined using long-duration parameters of intracortical microstimulation (LD-ICMS) based on area and spatial coordinates of separate motor output types to build upon our recent report in uninjured mice. LD-ICMS test sites that elicited complex (multi-joint) movement, simple (single skeletal joint) movement, as well as co-elicited FORELIMB + HINDLIMB responses were detected and recorded. Forelimb reaching behavior was assessed using the single pellet reaching (SPR) task. At 6 weeks post-surgery, behavioral deficits persisted and neocortical territories for separate movements exhibited differences in neocortical area, and spatial location, and differed between MCAo-Injured animals (i.e., the MCAo group) and Sham-Injured animals (i.e., the Control group). MCAo-Injury reduced neocortical area of complex movements while increasing area of simple movements. Limited effects of injury were detected for spatial coordinates of neocortical movements. Significant positive correlations were detected between final SPR performance and either area of complex retract or area of co-occurring FORELIMB + HINDLIMB sites.

Effect of early Sanguinate (PEGylated carboxyhemoglobin bovine) infusion on cerebral blood flow to the ischemic core in experimental middle cerebral artery occlusion

BackgroundSanguinate, a bovine PEGylated carboxyhemoglobin-based oxygen carrier with vasodilatory, oncotic and anti-inflammatory properties designed to release oxygen in hypoxic tissue, was tested to determine if it improves infarct volume, collateral...

Abstract P534: Relationship Between rCBF and Pial Collateral Recruitment in Experimental Acute Ischemic Stroke

Purpose: The purpose of this study was to assess the relationship between relative cerebral blood flow (rCBF) by MRI and leptomeningeal collateral recruitment in an experimental acute ischemic stroke model. Methods: This study was approved by the local IACUC. Eight mongrel canines (20-30kg) underwent endovascular middle cerebral artery occlusion (MCAO). Anesthetics were chosen not to interfere with cerebrovascular reactivity. Physiologic parameters were maintained within normal ranges. Pial collateral recruitment was angiographically assessed using a previously published 11-point scale. After the MCAO, subjects underwent DSC and DTI MRI (3T Achieva, Philips Healthcare, Best, Netherlands) in order to derive rCBF and infarct volume by diffusion restriction on mean diffusivity maps. DSC images were imported into Visage software (Visage Imaging; Richmond Australia). The region of interest was drawn on the penumbra and infarct core and corresponding contralateral side. Infarct volume was calculated by using a quantitative voxel-wise threshold by setting a threshold of 1.5 SD relative to normal values based on an ROI drawn to cover the entire contralateral normal hemisphere exclusive of the ventricles. Results: The pial collateral recruitment was strongly correlated with the rCBF on the penumbral region (r 2 = 0.7823, P-value= 0.003), and with the infarct volume measured two hours following occlusion (r 2 = 0.933376, P value= 0.00009.). There was a poor correlation between the pial collateral recruitment and the rCBF ratio on the core region (R 2 =0.4447, p-value= 0.07.) Conclusion: Preliminary data from this study suggests rCBF from the penumbral regions in experimental MCAO has a closer association with pial collateral recruitment relative to rCBF in the core infarct zone. This information has the potential to affect the interpretation of angiographic studies in acute ischemic stroke and underscores the importance of assessing regional differences in rCBF.

Abstract WP160: Conivaptan (Combined Vasopressin Receptor Antagonism) Attenuates Cerebral Edema Following Ischemic Stroke in Rodent Model

Objective: To study the potential role of Conivaptan on cerebral edema, infarct volume, neurological deficit and survival. Introduction: Although vasopressin has a significant detrimental role in stroke-related cerebral edema, the effect of mixed vasopressin antagonism in stroke has not been well studied. We studied the effects of conivaptan on the course of cerebral edema, severity of neurological deficit, infarct volume, aquaporin-4 (AQP4) protein levels and survival after experimental stroke. Methods: Animals were randomized to receive either conivaptan or vehicle after reperfusion of experimental middle cerebral artery occlusion. The severity of neurological deficit, edema, and infarct volume assessments were performed by an investigator blinded to group assignment. All assessments were performed at either 12h or 24h and Western blot was subsequently used to investigate AQP4 levels. Results: At 12h, conivaptan-treated mice (n=16) had 6.64±6.50% ipsilateral hemispheric enlargement compared to 16.55±7.05% in control mice (n=16, p=0.0003). Similarly, at 24h, conivaptan-treated mice (n=12) had 6.81±4.63% ipsilateral hemispheric enlargement in comparison to 13.93±5.43% in control mice (n=12, p=0.0023). At 24h, the conivaptan-treated mice had lower neurological deficits in comparison to control (p=0.04). There was no significant effect of conivaptan on infarct size or AQP4 levels in comparison to vehicle, naïve and sham group. Conclusions: The present study highlights the role of mixed vasopressin receptor antagonism in reducing cerebral edema secondary to brain ischemia. This data suggests the possibility of developing vasopressin antagonism as a new adjuvant in treatment of stroke-related brain edema.

Endothelial edema precedes blood-brain barrier breakdown in early time points after experimental focal cerebral ischemia

In the setting of stroke, ischemia-related blood-brain barrier (BBB) dysfunction aggravates the cerebral edema, which critically impacts on the clinical outcome. Further, an impaired vascular integrity is associated with the risk of intracranial bleeding, especially after therapeutic recanalization. Therefore, the present study was aimed to investigate early vascular alterations from 30 min to 4 h after experimental middle cerebral artery occlusion (MCAO) in mice. Here, an extravasation of the permeability marker FITC-albumin was detectable in animals 2 and 4 h after MCAO. Thereby, BBB breakdown correlated with alterations of the endothelial surface, indicated by a discontinuous isolectin-B4 staining, while tight junction strands remained detectable using electron and immunofluorescence microscopy. Noteworthy, already 30 min after MCAO, up to 60% of the ischemia-affected vessels showed an endothelial edema, paralleled by edematous astrocytic endfeet, clearly preceding FITC-albumin extravasation. With increasing ischemic periods, scores of vascular damage significantly increased with up to 60% of the striatal vessels showing loss of endothelial integrity. Remarkably, comparison of permanent and transient ischemia did not provide significant differences 4 h after ischemia induction. As these degenerations also involved penumbral areas of potentially salvageable tissue, adjuvant approaches of endothelial protection may help to reduce the vasogenic edema after ischemic stroke.

Why microglia kill neurons after neural disorders? The friendly fire hypothesis.

Neuroinflammation plays a fundamental role on the pathophysiology of acute and chronic neural disorders. Microglia activation is a major event following central nervous system inflammation displaying different phenotypes with beneficial and detrimental actions (a Janus face). The reason for this apparent duality is unknown. We have previously shown that following experimental middle cerebral artery occlusion in the rat brain, microglia seem to support and impair adult neurogenesis in the same ischemic striatum. Based on these results, we raised the hypothesis that in the same pathologic environment, gradients of different ligands distributed over different anatomical niches might contribute to both detrimental and beneficial microglial phenotypes. These ligands (“danger signals”) are released by dying cells and bind to microglial receptors in their membranes. Activation of different microglial receptors induces downstream biochemical pathways culminating in a spectrum of microglial phenotypes like M1 and M2 and others. In this paper, we first review the immune functions of microglia and the role of toll-like receptors on the fight against infections. We then briefly revise the dual role of microglia after neural disorders. We then propose a novel hypothesis to explain the Janus face of microglia during the pathophysiology of central nervous system diseases: the “friendly fire hypothesis”. According to this idea “danger signals” or danger associated molecular patterns released by stressed, damaged and/or dying cells during stroke, trauma and other diseases might activate microglial pattern-recognition receptors (i.e., toll like receptors) or other unidentified receptors normally activated by pathogens. This could activate the same genetic and biochemical machinery used by microglia to fight against pathogens even in the absence of infection. According to this notion, microglia may cause bystander neuronal damage with a kind of blind “friendly fire”, fighting against a non-existing infection during non-infectious disorders, like stroke and trauma. The “friendly fire hypothesis” is a novel proposal to explain why microglia may be detrimental and beneficial after acute and chronic neural disorders and may direct future investigations for developing of neuroprotective agents.

Abstract WP156: A Phase IIa Double-blind, Placebo Controlled Study of Extended-release Niacin for Stroke Recovery

Introduction: In pre-clinical studies, extended-release niacin has been shown to increase neurogenesis, synaptogenesis, and angiogenesis in a rat model of experimental middle cerebral artery (MCA) occlusion. Using this animal model, extended-release niacin has been shown to improve functional outcomes in rats when compared to placebo, beginning 24 hours after experimental stroke. Methods: This was a phase IIa double-blind, randomized, placebo controlled study (Henry Ford Health System funded; clinicaltrials.gov NCT00796887). At a single site (Henry Ford Hospital, Detroit, MI), patients with ischemic stroke 3 to 7 days prior and NIHSS 4-20 were randomized to two doses of oral extended-release niacin (500 mg or 1000 mg daily) or placebo. Primary outcome measures were change in mRS or NIHSS from baseline to Week 24. Results: There were 27 patients enrolled in this study. Ten patients were randomized to extended-release niacin 500 mg, 7 to 1000mg, and 10 to placebo. No significant differences were seen in baseline characteristics of enrolled patients. Significant increases in HDL-C were seen from baseline to 24 weeks among extended-release niacin treated patients (12.5 mg/dL, p=0.035), but not in placebo-treated patients (6.2 mg/dL, p=0.157). Mean improvement in NIHSS from baseline to 24 weeks of treatment was similar for both extended-release niacin and placebo treated patients (6.5 vs. 6.1, p=0.845). No differences were seen in functional outcome (mRS) at 24 weeks between extended-release niacin and placebo treated patients (2.1 vs. 2.0, p=0.860). There were a total of 40 AEs, with 27 in the extended-release niacin group and 10 in the placebo group. For these AEs, no clear interaction with treatment group could be confirmed. Conclusions: Extended-release niacin administered for 24 weeks starting between 3 and 7 days after ischemic stroke demonstrated no improvement in functional outcome as compared to placebo. Extended-release niacin was overall well tolerated. Although this small exploratory study did not identify a signal for potential efficacy, useful sample size estimates were gleaned. Any future clinical investigations may benefit from the collection of additional clinical measures of motor or gait function to differentiate treatment effects.

Increased functional connectivity after stroke correlates with behavioral scores in non-human primate model

Here we characterized the functional connectivity (FC) changes occurring after a controlled MCA stroke in a primate model. We hypothesize that if FC can inform about the neural changes after a stroke in the non-human primate (NHP) stroke model, then significant FC changes after the stroke would have to correlate with the remaining behavioral capacities. Eleven cynomolgus monkeys underwent an experimental middle cerebral artery occlusion while five monkeys remained as the control group. One month later the neurological function was assessed with a set of fine motor tasks and the Nonhuman Primate Stroke Scale (NHPSS). Structural and functional connectivity analyses were done to compare both groups. Three FC changes showed significant behavioral correlations: right sensorimotor-right lateral intraparietal FC with the six-well task; left posterior intraparietal-left dorsal premotor FC with the hill task; and right visual-left primary motor FC with the NHPSS. In the three instances, stronger FC correlated with better behavioral outcome. The results show that the functional changes correlating with behavioral outcomes involved sensorimotor cortices that were not restricted to the affected hemisphere. These results show that the FC analysis in NHP stroke model is a relevant methodology suitable to inform the neural changes occurring after a stroke.

Intracranial Pressure Elevation 24 h after Ischemic Stroke in Aged Rats Is Prevented by Early, Short Hypothermia Treatment.

Stroke is predominantly a senescent disease, yet most preclinical studies investigate treatment in young animals. We recently demonstrated that short-duration hypothermia-treatment completely prevented the dramatic intracranial pressure (ICP) rise seen post-stroke in young rats. Here, our aim was to investigate whether a similar ICP rise occurs in aged rats and to determine whether short-duration hypothermia is an effective treatment in aged animals. Experimental middle cerebral artery occlusion (MCAo-3 h occlusion) was performed on male Wistar rats aged 19–20 months. At 1 h after stroke-onset, rats were randomized to 2.5 h hypothermia-treatment (32.5°C) or normothermia (37°C). ICP was monitored at baseline, for 3.5 h post-occlusion, and at 24 h post-stroke. Infarct and edema volumes were calculated from histology. Baseline pre-stroke ICP was 11.2 ± 3.3 mmHg across all animals. Twenty-four hours post-stroke, ICP was significantly higher in normothermic animals compared to hypothermia-treated animals (27.4 ± 18.2 mmHg vs. 8.0 ± 5.0 mmHg, p = 0.03). Infarct and edema volumes were not significantly different between groups. These data demonstrate ICP may also increase 24 h post-stroke in aged rats, and that short-duration hypothermia treatment has a profound and sustained preventative effect. These findings may have important implications for the use of hypothermia in clinical trials of aged stroke patients.

The temporal evolution of diffusional kurtosis imaging in an experimental middle cerebral artery occlusion (MCAO) model

PurposeDiffusional kurtosis imaging (DKI), as a non-Gaussian diffusion model, has been applied in human and animal studies of ischemic stroke. This study aimed to intensively characterize the temporal evolution of DKI-derived variables in an experimental middle cerebral artery occlusion (MCAO) model, and to explore its potential application in ischemic stroke. Materials and methodsEleven MCAO rats and ten control rats underwent DKI and diffusion weighted imaging (DWI) scans, at different time points of 0.5, 2, 6, 12, 24 and 72h after operation respectively. The infarct area in DKI- and DWI-derived variables was compared among different time points, and between different groups [INFARCTION, MIRROR, CONTROL-R (right side of the control group, the same side as in the infarction group), CONTROL-L (left side of the control group)] using repeated measures analysis of variance (ANOVA). The percent changes from normal to that in ischemic tissues and histology were also evaluated. ResultsIn the infarct region, from 0.5 to 72h, MK, K∥, K⊥ demonstrated irregular high signal, whereas relative homogeneous low signals were revealed by MD, D∥, D⊥ and ADC. Compared with the MIRROR and CONTROL-R group, MK, K∥, K⊥ in the infarcted area increased aggressively which peaked at 12h and gradually decreased; MD, D∥, D⊥ and ADC decreased gradually until 12h and then began to increase gradually; FA decreased rapidly from 0.5 to 72h. MD, D∥ and ADC were significantly different between Mirror and CONTROL-L group (P<0.05). ConclusionDKI can provide more detailed information to describe ischemic lesion, and has great potential application in ischemic stroke.

Intracranial Pressure and Collateral Blood Flow.

Leptomeningeal collateral vessels, linking the 3 major arterial territories over the surface of the brain, have been recognized for >140 years.1 More widespread use of advanced clinical imaging in the past decade has led to increasing recognition of the key importance of collaterals in ischemic stroke outcome.2 However, recent studies from several groups indicate that failure of initially good collateral supply is a key feature of patients with delayed infarct expansion.3,4 This clinically challenging problem typically occurs in the first 1 to 2 days after hospital admission in patients with initially mild stroke symptoms. Rethrombosis of reperfused vessels was previously thought to be the likely cause of delayed infarct expansion in most patients. However, this theory is not supported by more recent evidence from imaging studies. Despite the important recent observations, there is limited understanding of the dynamic control of the collateral circulation, in particular, the cause of collateral blood flow failure. In this article, we will discuss recent observations from our experimental stroke model, indicating a dramatic increase in intracranial pressure (ICP) occurring around 24 hours after onset of even small stroke.5,6 We have also shown a significant linear reduction of collateral blood flow in response to progressive ICP elevation.7 We believe that a similar transient ICP elevation occurring during the first 1 to 2 days post stroke is a likely mechanism to explain delayed infarct expansion in patients with minor stroke. Perhaps surprisingly, we can find no published data on ICP at 24 hours in patients with minor stroke. The preclinical findings suggest that gathering such data should be a priority. ### Human Stroke There is a strong association between the extent of leptomeningeal collaterals and clinical stroke outcome. Initial studies using digital subtraction angiography permitted direct visualization of collateral vessels and …

Scutellarin regulates the Notch pathway and affects the migration and morphological transformation of activated microglia in experimentally induced cerebral ischemia in rats and in activated BV-2 microglia.

BackgroundActivated microglial cells release an excess of inflammatory mediators after an ischemic stroke. We reported previously that scutellarin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to middle cerebral artery occlusion (MCAO); however, the mechanism via which scutellarin exerts its effects on microglial activation has not been explored. This study aimed to elucidate if scutellarin can regulate the Notch pathway that is linked to microglia activation in MCAO rat, and in lipopolysaccharide (LPS)-induced BV-2 microglia. Along with this, we also investigated some characteristic behavioral responses of activated microglia.MethodsExpression of various members of the Notch pathway, including Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK) and transcription factor hairy and enhancer of split-1 (Hes-1) in activated microglia was assessed by immunofluorescence staining and western blot after experimental MCAO and in vitro LPS activation. The effect of scutellarin on migration of microglia was determined by the transwell chamber assay as well as expression of monocyte chemoattractant protein-1 (MCP-1). The morphological change of microglia induced by scutellarin was detected by F-actin staining and electron microscopy.ResultsScutellarin markedly attenuated the expression of NF-κB, Notch-1, NICD, RBP-JK and Hes-1 both in vivo and in activated microglia. It decreased the expression of MCP-1 and microglial migration, but increased the ability of microglia adhesion. Scutellarin also altered the phenotype of microglia by causing rearrangement or reorganization of its cytoskeleton.ConclusionsThe results suggest that scutellarin regulates the activation of microglia via the Notch pathway and concurrently induces morphological and functional changes in activated microglia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0226-z) contains supplementary material, which is available to authorized users.

Monitoring Bone Marrow Stem Cells with a Reporter Gene System in Experimental Middle Cerebral Artery Occlusion Rat Models

This study was designed to investigate the feasibility of imaging bone marrow stem cells (BMSCs) in experimental middle cerebral artery occlusion (MCAO) rat models with a reporter gene-probe system, HSV1-tk-(131)I-2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ((131)I-FIAU), and to choose the best strategies for stem cell injection, image acquisition, and imaging in vivo. A recombinant adenovirus (Ad5-TIBE) carrying the herpes simplex virus type 1 thymidine kinase (TK) reporter gene (HSV1-tk) linked via the internal ribosome entry site to the brain-derived neurotrophic factor therapeutic gene was prepared. After transfection with Ad5-TIBE, BMSCs were introduced into MCAO rat models via local injection into the brain or via injection into the lateral ventricle, carotid artery, and tail vein. Normal rats were used as controls. Twenty-four hours after (131)I-FIAU injection, rats were sacrificed for biodistribution analysis. The expression of the TK gene was evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. Autoradiography was used for ex vivo imaging. SPECT images were obtained in MCAO rat models. The percentage injected dose per gram (%ID/g) in infarcted brain tissue in rats receiving the injection into the brain was 0.124 ± 0.013; this value was significantly higher than those in rats receiving the injection into the ventricle (0.052 ± 0.004), carotid artery (0.061 ± 0.002), and tail vein (0.059 ± 0.005) as well as normal rats (0.005 ± 0.001). No differences were seen in the other cell transplantation groups. The %ID/g in infarcted brain tissue was higher than that in the contralateral brain tissue in all experimental rats but not in normal rats. The expression of the TK gene in rats receiving a local injection into the brain was superior to that in all of the other groups. TK messenger RNA and protein expression showed a positive correlation with the %ID/g in brain tissue. Greater radioactivity at the injection site than in the surrounding and contralateral brain tissues in all experimental rats was indicated through autoradiography. The ratio of counts in bilateral brain tissues reached its peak (6.63) 24 h after (131)I-FIAU injection. SPECT images showed that radioactivity accumulation in the brain was low but increased gradually over time. The HSV1-tk-(131)I-FIAU reporter gene-probe system may be used to monitor BMSC activity in experimental MCAO rat models. Local injection of stem cells may provide an optimal means for cell transplantation, and imaging with (131)I-FIAU 24 h after injection provides peak target-to-nontarget count ratios.

Abstract 3842: Angiographic Assessment Of Leptomeningeal Collateral Perfusion Parameters Following Experimental Transient Middle Cerebral Artery Occlusion

Objectives: This work aims to develop and validate an angiographically based quantitative assessment of leptomeningeal collateral perfusion (QLCP) in experimental reversible middle cerebral artery occlusion (MCAO). Methods: Pial collaterals were assessed during MCAO using an angiographically based transient MCAO model in eight mongrel dogs (20-30 kg). Angiographic images were analyzed using a custom-made MATLAB program which measured contrast density over time. Using bivariate linear fit analysis relative cerebral blood volume (rCBV), relative transit time (rTT) and relative cerebral blood flow (rCBF) derived from regions of interest (ROI) from the normal and abnormal hemispheres were extracted and compared to one hour post reperfusion MRI based infarct volume calculations and leptomeningeal collateral scoring using previously published methods. Results: QLCP was reproducibly assessed but variably predictive of infarct volume on one hour post reperfusion mean diffusivity maps using rCBV (p&lt;0.0001; r2=0.937), rTT (p = 0.05, r2 = 0.494), and rCBF (p=0.0024, r2=0.807). Leptomeningeal collateral scoring variably correlated with rCBV (p&lt;0.0001, r2 = 0.948), rTT (p=0.0285, r2= 0.578) and rCBF (p0.0021, r2= 0.817). Conclusion: QLCP was validated in an experimental MCAO model based on correlation with a leptomeningeal collateral scoring system. QLCP assessment of rCBV is a better predictor for infarct volume than rTT or rCBF in a transient MCAO model. It is noteworthy that an angiographically based assessment of rCBV, rTT and rCBF differs from CT and MRI based assessments. In particular , the time frame used and the relative density of the vasculature on the derived color maps differ ( figure 1). figure 1: QLCP derived cerebral blood volume map. The boxes indicate regions of interest for analysis.

Neuroserpin Protects Neurons from Ischemia-Induced Plasmin-Mediated Cell Death Independently of Tissue-Type Plasminogen Activator Inhibition

The serine proteinase tissue-type plasminogen activator (tPA) and the serine proteinase inhibitor neuroserpin are both expressed in areas of the brain with the highest vulnerability to hypoxia/ischemia. In vitro studies show that neuroserpin inhibits tPA and, to a lesser extent, urokinase-type plasminogen activator and plasmin. Experimental middle cerebral artery occlusion (MCAO) increases tPA activity and neuroserpin expression in ischemic tissue, and genetic deficiency of tPA or either treatment with or overexpression of neuroserpin decreases the volume of the ischemic lesion following MCAO. These findings have led to the hypothesis that neuroserpin's neuroprotection is mediated by inhibition of tPA's alleged neurotoxic effect. Ischemic preconditioning is a natural adaptive process whereby exposure to a sublethal insult induces tolerance against a subsequent lethal ischemic injury. Here we demonstrate that exposure to sublethal hypoxia/ischemia increases the neuroserpin expression in the hippocampal CA1 layer and cerebral cortex, and that neuroserpin induces ischemic tolerance and decreases the volume of the ischemic lesion following MCAO in wild-type and tPA-deficient (tPA-/-) neurons and mice. Plasmin induces neuronal death, and this effect is abrogated by either neuroserpin or the NMDA receptor antagonist MK-801. Neuroserpin also attenuated kainic acid-induced neuronal death. Our data indicate that the neuroprotective effect of neuroserpin is due to inhibition of plasmin-mediated excitotoxin-induced cell death and is independent of neuroserpin's ability to inhibit tPA activity.

Tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 mediate cerebral ischemia-induced poly(ADP-ribose) polymerase-1 activation and neuronal death

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Fn14) are expressed in neurons. Here we demonstrate that TWEAK induces a dose-dependent increase in neuronal death and that this effect is independent of tumor necrosis factor alpha (TNF-α) and mediated by nuclear factor-kappa B (NF-κB) pathway activation. Incubation with TWEAK induces apoptotic cell death in wild-type (Wt) but not in Fn14 deficient (Fn14 −/−) neurons. Intracerebral injection of TWEAK induces accumulation of poly(ADP-ribose) polymers (PAR) in Wt but not in Fn14 −/− mice. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in Wt neurons, and decreases cell survival in Wt but not in Fn14 −/− or TWEAK deficient (TWEAK −/−) neurons. Experimental middle cerebral artery occlusion (MCAO) increases the expression of TWEAK and Fn14 mRNA and active caspase-3, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) with accumulation of PAR in the ischemic area in Wt but not Fn14 −/− mice. Together, these results suggest a model where in response to hypoxia/ischemia the interaction between TWEAK and Fn14 in neurons induces PARP-1 activation with accumulation of PAR polymers and cell death via NF-κB pathway activation. This is a novel pathway for hypoxia/ischemia-induced TWEAK-mediated cell death and a potential therapeutic target for ischemic stroke.

Interleukins and Ischemic Stroke: Effect of Neutralization of Rat IL-6 Bioactivity on the Degree of Ischemic Cerebral Damage Caused by Experimental Middle Cerebral Artery Occlusion in the Rat

The purpose was to investigate the effect of neutralization of rat interleukin-6 (IL-6) bioactivity on the activity levels of Na+-K+/Mg++ adenosine-5′-triphosphatase and superoxide dismutase, and the degree of infarct size in ischemic brain produced by experimental middle cerebral artery occlusion in the rat. Seventy adult male Wistar rats weighing 280 to 310 g were used in this study. The animals were randomly allocated to groups: group I (n=10) consisted of normal controls, group II (sham-operated, n=20) underwent surgical exposure of the middle cerebral artery but no cauterization, and the remaining 40 rats were subjected to middle cerebral artery occlusion. Immediately after occlusion, each of these 40 rats was randomly assigned to either the occlusion-only group (group III, n=20) or the occlusion plus IL-6 antibody treatment group (group IV, n=20). All evaluations were performed in blinded fashion. Our data provide evidence that neutralization of IL-6 bioactivity correlates with less secondary damage in long-term treatment after focal cerebral ischemia in the rat.

Detrimental Effects of 60 kHz Sonothrombolysis in Rats with Middle Cerebral Artery Occlusion

Recent studies have raised concerns about the safety of low frequency ultrasound in transcranial therapeutic application in cerebral ischemia. This study was designed to evaluate safety aspects and potential deleterious effects of low frequency, 60 kHz ultrasound in treatment of experimental middle cerebral artery occlusion (MCAO) in rats. Forty-five male Wistar rats were submitted to either temporary (90 min; groups I and II) or permanent MCAO (groups III and IV) using the suture technique. All animals received recombinant tissue plasminogen activator (rt-PA) starting 90 min after the beginning of occlusion. Groups I and III were additionally treated with 60 kHz ultrasound (time average acoustic intensity 0.14 W/cm 2, duty cycle 50%). Outcome assessment consisted of magnetic resonance imaging (MRI) and clinical evaluation after 5 and 24 h, and histology (perfusion fixation after 24 h). Overall mortality was higher in animals treated with ultrasound (43% versus 29% in controls). Most animals died during the insonation period (25% in group I, 36% in group III, no animals in the corresponding control groups; p < 0.05). Histology revealed disseminated microscopic intracerebral bleeding and subarachnoid hemorrhage as one possible cause of death. After temporary occlusion, the hemispheric ischemic lesion volume was more than doubled in animals treated with ultrasound (20.3% ± 14.1% versus 8.6% ± 5.1% in controls; p < 0.05). No difference in lesion volume was seen after permanent MCAO. Neurological assessment showed impairment of hearing as an additional specific side effect in ultrasound treated animals (65%, no impairment in controls). Although the results are not directly transferable to the human setting, this study clearly demonstrates the potential limitations of low frequency therapeutic ultrasound and the importance of pre-clinical safety assessment. (E-mail: max.nedelmann@neuro.med.uni-giessen.de)

Vasoconstrictive Neurovascular Coupling during Focal Ischemic Depolarizations

Ischemic depolarizing events, such as repetitive spontaneous periinfarct spreading depolarizations (PIDs), expand the infarct size after experimental middle cerebral artery (MCA) occlusion. This worsening may result from increased metabolic demand, exacerbating the mismatch between cerebral blood flow (CBF) and metabolism. Here, we present data showing that anoxic depolarization (AD) and PIDs caused vasoconstriction and abruptly reduced CBF in the ischemic cortex in a distal MCA occlusion model in mice. This reduction in CBF during AD increased the area of cortex with 20% or less residual CBF by 140%. With each subsequent PID, this area expanded by an additional 19%. Drugs that are known to inhibit cortical spreading depression (CSD), such as N-methyl-D-aspartate receptor antagonists MK-801 and 7-chlorokynurenic acid, and sigma-1 receptor agonists dextromethorphan and carbetapentane, did not reduce the frequency of PIDs, but did diminish the severity of episodic hypoperfusions, and prevented the expansion of severely hypoperfused cortex, thus improving CBF during 90 mins of acute focal ischemia. In contrast, AMPA receptor antagonist NBQX, which does not inhibit CSD, did not impact the deterioration in CBF. When measured 24 h after distal MCA occlusion, infarct size was reduced by MK-801, but not by NBQX. Our results suggest that AD and PIDs expand the CBF deficit, and by so doing negatively impact lesion development in ischemic mouse brain. Mitigating the vasoconstrictive neurovascular coupling during intense ischemic depolarizations may provide a novel hemodynamic mechanism of neuroprotection by inhibitors of CSD.

Effect of neutralization of rat IL-6 bioactivity on collateral blood supply from retrograde flow via cortical anastomoses in the rat central nervous system

The purpose of this study was to determine whether neutralization of rat interleukin-6 (IL-6) bioactivity increases the collateral blood supply from retrograde flow via the major middle cerebral artery branches after experimental middle cerebral artery occlusion in the rat. Seventy rats were randomly allocated to four main groups: Group I (n = 10) consisted of normal controls; Group II (n = 20) underwent craniectomy only; Group III (n = 20) was subjected to middle cerebral artery occlusion; and Group IV (n = 20) underwent middle cerebral artery occlusion and treatment with anti-rat IL-6 antibody. Half of the rats from each of Groups II, III and IV were killed at 24 h and the other half at 72 h after craniectomy alone or occlusion. A single dose of antibody did not affect middle cerebral artery caliber, but administration of three doses resulted in a significant increase in the diameter of middle cerebral artery compared to the findings in the corresponding occlusion-only groups. The results suggest that neutralization of rat IL-6 bioactivity in longterm recovery increases the collateral blood supply from retrograde flow via cortical anastomoses after experimental arterial occlusion in the rat brain. [Neurol Res 2002; 24: 405-408]