Permanent Middle Cerebral Artery Occlusion Model Research Articles

Dynamic assessment of brain perfusion in a middle cerebral artery occlusion rat model by contrast-enhanced ultrasound imaging: a pilot study.

The middle cerebral artery occlusion model (MCAo) is a commonly used animal model for cerebral ischemia studies but lacks accessible imaging techniques for the assessment of hemodynamic changes of the model. The study aims to explore the value of contrast-enhanced ultrasound (CEUS) in evaluating brain perfusion in the early stages after MCAo surgery. In total, 18 adult male Sprague-Dawley rats were subjected to right MCAo using an intraluminal filament model, and CEUS was performed at the three following timepoints: before (T0), immediately after (T1), and 6 h after permanent MCAo (T2). Twelve rats successfully completed the study, and their brains were removed and stained using 2, 3, 5-triphenyltetrazolium chloride (TTC). CEUS video images were visualized offline, and the time-intensity curves (TICs) were analyzed. Different cerebrovascular patterns and manifestations of the contrast enhancement in rat ischemic hemispheres were observed. Semi-quantitative parameters of TICs in ischemic areas (ROIi) and the surrounding normal- or hypo-perfused areas (ROIn) were calculated and compared between T0, T1, and T2, and also between ROIi and ROIn. A significant correlation was found between the lesion volume (%) determined by TTC and CEUS parameters (r = -0.691, P = 0.013 for peak intensity; r = -0.742, P = 0.006 for area under the curve) at T2. After the same occlusion, there were differences in contrast perfusion in each group. This study suggests that CEUS could be an effective imaging tool for studying cerebral ischemia and perfusion in small animals as long as the transcranial acoustic window allows it.

Protective effect and mechanism of Ershiwuwei Zhenzhu Pills on cerebral apoplexy rats

To study the protective effect of Ershiwuwei Zhenzhu Pills on ischemic stroke rats. Ninety 4-weeks-old SPF male SD rats were randomly divided into 6 groups(n=15):sham operation group, model group, nimodipine group(12 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills high-dose group(400 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills medium-dose group(200 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills low-dose group(100 mg·kg~(-1)).The permanent middle cerebral artery occlusion model(PMCAO) was established in the model group, nimodipine group, and Ershiwuwei Zhenzhu Pills groups by the improved thread plug method, while the sham operation group did not insert the thread plug.Nimodipine group and Ershiwuwei Zhenzhu Pills groups were given intragastric administration once a day for 24 days before the modeling operation, and once 1 hour before the modeling operation, while sham operation group and model group were given equal volumes of distilled water.The neuroethology of the surviving rats was measured; The volume of cerebral infarction in rats was measured by TTC method; The histopathology of rat brain was observed by HE method; The expression levels of tumor necrosis factor α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),malondialdehyde(MDA),superoxide dismutase(SOD) and catalase(CAT) in serum were detected by ELISA;The mRNA expressions of Notch 1,Jagged 1,Hes 1 and Bcl-2 in rat brain were detected by RT-PCR;Western blot was used to detect the expression levels of caspase-3 protein in rat brain; the expression levels of vascular endothelial growth factor(VEGF) and CD34 positive cells in rat brain were detected by immunofluorescence.The low, medium and high dose groups of Ershiwuwei Zhenzhu Pills and nimodipine group could significantly reduce the neurobehavioral score and cerebral infarction volume of rats with permanent middle cerebral artery occlusion, reduce the morphological changes of nerve cells, decrease the expression of TNF-α,IL-1β and IL-6 in rat serum, increase the activity of SOD and CAT,and reduce the level of MDA.Furthermore, the expression levels of Notch l, Jagged l, Hes l and Bcl-2 mRNA were significantly increased, and the expression level of caspase-3 protein was decreased.Meanwhile, the number of VEGF and CD34 positive cells increased in the treatment group.The differences were statistically significant. Ershiwuwei Zhenzhu Pills has a protective effect on ischemic stroke rats, and its mechanism may be related to anti-inflammation, anti-oxidation, promotion of nerve cell proliferation, inhibition nerve cell apoptosis and promotion of angiogenesis.

Delayed recanalization after MCAO ameliorates ischemic stroke by inhibiting apoptosis via HGF/c-Met/STAT3/Bcl-2 pathway in rats

The activation of tyrosine kinase receptor c-Met by hepatocyte growth factor (HGF) showed an anti-apoptotic effect in numerous disease models. This study aimed to investigate the neuroprotective mechanism of the HGF/c-Met axis-mediated anti-apoptosis underlying the delayed recanalization in a rat model of middle cerebral artery occlusion (MCAO). Permanent MCAO model (pMCAO) was induced by intravascular filament insertion. Recanalization was induced by withdrawing the filament at 3 days after MCAO (rMCAO). HGF levels in the blood serum and brain tissue expressions of HGF, c-Met, phosphorylated-STAT3 (p-STAT3), STAT3, Bcl-2, Bax, cleaved caspase-3(CC3) were assessed using ELISA and western blot, respectively. To study the mechanism, HGF small interfering ribonucleic acid (siRNA) and c-Met inhibitor, su11274, were administered intracerebroventricularly (i.c.v.) or intranasally, respectively. The concentration of HGF in the serum was increased significantly after MCAO. Brain expression of HGF was increased after MCAO and peaked at 3 days after recanalization. HGF and c-Met were both co-localized with neurons. Compared to rats received permanent MCAO, delayed recanalization after MCAO decreased the infarction volume, inhibited neuronal apoptosis, and improved neurobehavioral function, increased expressions of p-STAT3 and its downstream Bcl-2. Mechanistic studies indicated that HGF siRNA and su11274 reversed the neuroprotection including anti-apoptotic effects provided by delayed recanalization. In conclusion, the delayed recanalization after MCAO increased the expression of HGF in the brain, and reduced the infarction and neuronal apoptosis after MCAO, partly via the activation of the HGF/c-Met/STAT3/Bcl-2 signaling pathway. The delayed recanalization may serve as a therapeutic alternative for a subset of ischemic stroke patients.

Abstract TP120: Complement Activation Contributes to Malignant Transformation of Stroke and Limits Cognitive Recovery After Reperfusion Therapy

Introduction: Ischemic stroke involves an ischemic event followed by post-ischemic neuroinflammation, leading to acute mortality, neuronal loss, and impaired recovery. Standard of care reperfusion therapy (thrombolysis or thrombectomy) has a limited treatment window due to risk of hemorrhage, and leads to functional independence in only a subset of patients. We studied the role of complement and neuroinflammation in the pathophysiology behind the major clinical challenges of reperfusion therapy; the risk of hemorrhage and limited cognitive recovery. Methods: We used 3 stroke models, namely transient middle cerebral artery occlusion (MCAO), permanent MCAO and microembolic models, to investigate the effects of t-PA, mechanical reperfusion, rehabilitation therapy, and complement inhibition in mice. We used B4-Crry, a site-targeted inhibitor of C3 activation that specifically localizes to neoepitopes expressed in the post-ischemic brain following its intravenous administration. A retrospective analysis was also performed for cognitive and motor recovery in human patients following reperfusion therapy. Results: Complement inhibition with B4-Crry improved the safety, efficacy and treatment window of reperfusion therapy, decreased hemorrhagic transformation, and improved cognitive recovery. Despite reperfusion with t-PA, there was ongoing complement-dependent microglial activation and phagocytosis of hippocampal synapses for at least 30 days leading to pronounced long-term cognitive deficits. Administration of B4Crry, alone or in combination with thrombolytic therapy, limited complement deposition in the perilesional brain, and reduced microgliosis and synaptic uptake. Reperfusion therapy alone did not improve cognitive outcomes, even when combined with rehabilitation therapy. Reperfusion therapy did not improve cognitive outcomes in stroke patients. Conclusions: Although post-stroke reperfusion therapy limits the size of injury and improves motor deficits, post-reperfusion complement activation and neuroinflammation contribute to the malignant transformation of stroke acutely and seeds a chronic neurodegenerative inflammatory response. Complement modulation may be a promising adjuvant to reperfusion therapy.

Inhibiting 12/15-lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis models

12/15-Lipoxygenase (12/15-LOX) contributes to the brain damage after middle cerebral artery occlusion (MCAO) in the acute phase of stroke. The aim of this study was to investigate the effects of a 12/15-LOX inhibitor, LOXBlock-1(LB1), in mice using a FeCl3-induced permanent distal MCAO model and FeCl3-induced ischemia/thrombolysis with tPA. In order to induce permanent distal MCAO, 30% FeCl3 was used in C57BL6 mice. LB1 or DMSO treatments were applied intraperitoneally 2 h following MCAO. For FeCl3-induced ischemia/thrombolysis experiments, 10% FeCl3 was preferred so as to obtain reperfusion with tPA in CD1 mice. 4 h following ischemia either LB1 or DMSO and iv tPA was administered. Outcomes were NSS, weight loss, infarct volume, hemorrhage area and reperfusion rate. FeCl3-induced distal MCAO caused an increase in 12/15-LOX signal in the ischemic cortex with an increase in MDA2 and AIF immunoreactivity. LB1 treatment, applied 2 h after ischemia, significantly decreased the infarct volume at 24 h of permanent distal MCAO. Weight loss was also significantly reduced in LB1 treated group. Distal MCAO and tPA application with LB1 or DMSO showed that treatment significantly decreased the infarct volume and the hemorrhage area. The reperfusion rate in the LB1-treated group was surprisingly higher than in the DMSO group and NSS results were significantly improved. These data suggest that LB1 can be used as an adjuvant agent to tPA. This study not only shows the effects of LB1 treatment in distal MCAO but also confirms that FeCl3-induced MCAO model can be a useful tool to screen novel treatment options in stroke.

Abstract T P229: Deletion of Voltage Gated Proton Channel in Microglial Cells is Neurovascular Protective After Ischemic Brain Injury

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.

Identification of differentially expressed proteins and validation of the changes of N-ethylmaleimide-sensitive factor in rats with focal cerebral ischemia after transection of the cervical sympathetic trunk.

Stellate ganglion blockade (SGB) protects patients from focal cerebral ischemic injury, and transection of the cervical sympathetic trunk (TCST) in a rat model can mimic SGB in humans. The purpose of this study was to investigate the mechanisms underlying the neuroprotective effects of TCST on neuronal damage in the hippocampus in a rat model of middle cerebral artery occlusion (MCAO) in an attempt to elucidate the neuroprotective effects of SGB. The modified method of Zea Longa was used to establish the permanent MCAO model. Male Wistar rats were randomly divided into three groups: sham-operated group, MCAO group, and TCST group. The animals in TCST group were sacrificed 48 h after TCST which was performed after the establishment of the MCAO model. Proteins were extracted from the ipsilateral hippocampus and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and peptide mass fingerprinting (PMF). The levels of N-ethylmaleimide-sensitive factor (NSF) were measured as well. The results showed that 11 types of proteins were identified by 2D-DIGE. The expressions of eight proteins were changed both in the sham-operated and TCST groups, and the expressions of the other three proteins were changed in all three groups. Moreover, the expression of NSF was higher in the TCST group than in the MCAO group but lower in the MCAO group than in sham-operated group. The ratio of NSF expression between the MCAO group and shamoperated group was -1.37 (P<0.05), whereas that between the TCST group and MCAO group was 1.35 (P<0.05). Our results imply that TCST increases the expression of NSF in the hippocampus of adult rats with focal cerebral ischemia, which may contribute to the protection of the injured brain. Our study provides a theoretical basis for the therapeutic application of SGB to patients with permanent cerebral ischemia.

Efficacy of liposome-encapsulated hemoglobin in a rat model of cerebral ischemia.

Use of liposome-encapsulated hemoglobin (LEH) for oxygen delivery in the treatment of cerebral ischemia has been studied previously and its expected benefits confirmed. However, the relationship between the timing of administration and the efficacy of LEH in cerebral ischemia has not been studied in detail. We therefore investigated the therapeutic time window of LEH by using a rat model of cerebral ischemia, as well as evaluating the contribution of oxygen delivery to the efficacy of LEH. Dose-dependent effects and the therapeutic time window of LEH were studied using models of transient and permanent middle cerebral artery occlusion (MCAO), respectively, in SD rats. LEH was intravenously administered at 0.5 h after the onset of ischemia in the transient MCAO model and at 0.5, 2, 4, or 6 h in the permanent MCAO model. Efficacy of LEH treatment was evaluated using the infarct volume, which was examined with 2,3,5-triphenyltetrazolium chloride staining and estimated by integrating the unstained areas in serial sections of cerebral tissue. Effects of oxygen delivery by LEH were examined immunohistochemically with pimonidazole to stain for areas of low oxygen tension in the tissue. LEH treatment dose-dependently reduced the cerebral infarct volume, which was especially significant in the cortical region at doses of over 60 mg hemoglobin (Hb)/kg. In rats with permanent MCAO, LEH administration at a dose of 300 mg Hb/kg at 0.5 h and 2 h after the onset of cerebral ischemia significantly reduced cerebral infarct volume. Furthermore, immunohistochemical staining with pimonidazole showed that the areas of cerebral tissue that were hypoxic and had abnormal histological structure were reduced after LEH treatment. These results indicated that LEH is efficacious in the treatment of cerebral infarction secondary to MCAO and that oxygen delivery to ischemic cerebral tissues by LEH administered early after the onset of cerebral ischemia contributes to this effect.

Postischemic Brain Infiltration of Leukocyte Subpopulations Differs among Murine Permanent and Transient Focal Cerebral Ischemia Models

Cellular and humoral inflammations play important roles in ischemic brain injury. The effectiveness of immunomodulatory therapies may critically depend on the chosen experimental model. Our purpose was to compare the post-ischemic neuroinflammation among murine permanent and transient middle cerebral artery occlusion (MCAO) models. Permanent MCAO was induced by transtemporal electrocoagulation and 30 minutes or 90 minutes transient MCAO was induced by intraluminal filament in C57BL/6 mice. Infiltration of leukocyte subpopulations was quantified by immunohistochemistry and fluorescence-activated cell sorting. Cerebral cytokine and adhesion molecule expression was measured by real-time polymerase chain reaction (RT-PCR). Neutrophil infiltration was noted at 24 h after transient MCAO, but did not further increase until 5 days in the permanent MCAO model. Few T cells were observed in both MCAO models at 24 h, but permanent MCAO demonstrated much more infiltrating T cells at 5 days. Pronounced microglial activation was evident at 24 h and 5 days after permanent but not after transient MCAO. The number of invading NK cells and expression of MHCII on CD11b+ cells did not differ among the three groups. Five days after MCAO, the expression of IL-1, TNF-α and IFN-γ and of the adhesion molecules ICAM-1 and VCAM-1 was significantly higher in the permanent than in the transient MCAO groups. Cellular and humoral inflammation differs substantially among commonly used MCAO models. Neuroinflammation is more pronounced after permanent electrocoagulatory MCAO compared with 30 minutes and 90 minutes filament-MCAO.

Optimizing Suture Middle Cerebral Artery Occlusion Model in C57BL/6 Mice Circumvents Posterior Communicating Artery Dysplasia

The suture middle cerebral artery occlusion (MCAO) model is used worldwide in both academia and industry. However, the variable occurrence of dysplasia in posterior communicating arteries (PcomAs) induces high mortality and instability in permanent MCAO models, limiting the model's application to transient focal ischemia. In particular, high mortality in intraluminal suture MCAO models is associated with the dysplasia of PcomAs in C57BL/6 mice. Optimization of silicone coating length is critical for reducing mortality and generating stable infarct in this model. The aim of our study is to reduce mortality and improve the reproducibility of the intraluminal suture MCAO model in C57BL/6 mice, which have high variation in PcomA dysplasia. Adult male C57BL/6 mice (n=38) underwent MCAO using sutures with various diameters and silicone coating lengths. The occlusion of cerebral vessels was examined by synchrotron radiation live angiography. The morphology of PcomAs was examined under a microscope after MICROFIL(®) infusion. Neurological outcome, infarct volume, and mortality were examined within 28 days. Optimizing the silicone coating on an 8-0 suture tip, we were able to reduce the model mortality to zero after permanent occlusion in C57BL/6 and produce stable brain infarct volume independent of the patency of PcomAs.

Unraveling the ischemic brain transcriptome in a permanent middle cerebral artery occlusion mouse model by DNA microarray analysis

SUMMARYBrain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, leading to tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, as well as the underlying mechanisms of their action. The present study was initiated to investigate molecular responses at the level of gene expression in ischemic brain tissue. To achieve this, we used a mouse permanent middle cerebral artery occlusion (PMCAO) model in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, the right (ipsilateral) and left (contralateral) hemispheres of PMCAO model mice were dissected at two time points, 6 and 24 hours post-ischemia. Total RNA from the ischemic (ipsilateral) hemisphere was subjected to DNA microarray analysis on a mouse whole genome 4x44K DNA chip using a dye-swap approach. Functional categorization using the gene ontology (GO, MGD/AMIGO) of numerous changed genes revealed expression pattern changes in the major categories of cellular process, biological regulation, regulation of biological process, metabolic process and response to stimulus. Reverse-transcriptase PCR (RT-PCR) analysis on randomly selected highly up- or downregulated genes validated, in general, the microarray data. Using two time points for this analysis, major and minor trends in gene expression and/or functions were observed in relation to early- and late-response genes and differentially regulated genes that were further classified into specific pathways or disease states. We also examined the expression of these genes in the contralateral hemisphere, which suggested the presence of bilateral effects and/or differential regulation. This study provides the first ischemia-related transcriptome analysis of the mouse brain, laying a strong foundation for studies designed to elucidate the mechanisms regulating ischemia and to explore the neuroprotective effects of agents such as target neuropeptides.

Extension of the neuroprotective time window for thiazolidinediones in ischemic stroke is dependent on time of reperfusion

Stroke is a leading cause of death and disability but has limited therapeutic options. Thiazolidinediones (TZDs), agonists for the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)γ, reduce infarct volume and improve neurologic function following transient middle cerebral artery occlusion (MCAO) in rats. Translation of these findings into clinical therapy will require careful assessment of dosing paradigms and effective time windows for treatment. Understanding the mechanisms by which TZDs protect the brain provides insight into how time windows for neuroprotection might be extended. We find that two TZDs, pioglitazone and rosiglitazone, significantly reduce infarct volume at doses similar to those used clinically (1 mg/kg for pioglitazone and 0.1 mg/kg for rosiglitazone). We also find that pioglitazone reduces infarction volume in a transient, but not a permanent MCAO model suggesting that reperfusion plays an important role in TZD mediated neuroprotection. Since PPARγ agonists reduce inflammation and oxidative stress, both of which are exacerbated by reperfusion, we hypothesized that TZDs would be most effective if administered prior to reperfusion. We administered TZDs 3 h after MCAO and found that infarction volume and neurologic function are significantly improved in animals reperfused at 3 h and 15 min (after TZD treatment), but not in animals reperfused at 2 h (before TZD treatment) when assessed either 24 h or 3 weeks after MCAO. While TZDs reduce intercellular adhesion molecule (ICAM) expression to a similar extent regardless of the time of reperfusion, leukocyte entry into brain parenchyma is more dramatically reduced when reperfusion is delayed until after drug treatment. The finding that delaying reperfusion until after TZD treatment is beneficial despite a longer period of ischemia, is dramatic given the widely held view that duration of ischemia is the most important determinate of injury.

Therapeutic time window of mesenchymal stem cells derived from bone marrow after cerebral ischemia

Intravenous transplantation of mesenchymal stem cells (MSCs) prepared from bone marrow ameliorates functional deficits in rat cerebral infarction models. Although several hypotheses to account for these therapeutic effects have been suggested, angiogenesis is thought to be largely responsible. MSCs were intravenously infused into rats in the relatively later time points after permanent middle cerebral artery occlusion (MCAO) to determine if they could contribute to neovascularization and functional recovery. Although MRI analysis revealed that only rats receiving MSCs 7 days after MCAO exhibited decreased ischemic lesion volume than the control group, all MSCs treated rats receiving MSCs up to 1 month after MCAO had greater angiogenesis near the border of the ischemic lesions and showed greater functional recovery in the treadmill stress test than did control rats. Thus, these results suggest that the time window of MSC therapy is at least over 1 month after cerebral infarction in the rat permanent MCAO model, and systemic delivery of MSCs in the later phase after cerebral ischemia may have beneficial effect through an angiogenic mechanism.

Characterization of Gadolinium-Based Dynamic Susceptibility Contrast Perfusion Measurements in Permanent and Transient MCAO Models with Volumetric Based Validation by CASL

Perfusion imaging is crucial in imaging of ischemic stroke to determine 'tissue at risk' for infarction. In this study we compared the volumetric quantification of the perfusion deficit in two rat middle-cerebral-artery occlusion (MCAO) models using two gadolinium-based contrast agents (P1152 (Guerbet) and Magnevist (Bayer-Schering, Pittsburgh, PA, USA)) as compared with our well established continuous arterial spin labeling (CASL) perfusion imaging technique. Animals underwent either permanent MCAO or transient MCAO with 80-min reperfusion. Imaging was performed at four different time points after MCAO. A region-of-interest (ROI) analysis of the subregions of the ischemic zone (core, penumbra, transient reversal (TR), and sustained reversal (SR)) using P1152 showed significant reduction in blood flow in the core and TR subregions relative to the penumbral and SR subregions while occluded. After reperfusion, a significant increase in blood flow was recorded at all time points after reperfusion in all regions except TR. From the ROI analysis the threshold for the penumbra was determined to be -62+/-11% and this value was subsequently used for quantification of the volumetric deficit. The ischemic volume as defined by dynamic susceptibility contrast (DSC), was only statistically different from the CASL-derived ischemic volume when using Magnevist at post-reperfusion time points.

Protective Effect of Delayed Treatment With Low-Dose Glibenclamide in Three Models of Ischemic Stroke

Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied. We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride. Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling. Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.

Effect of large dose hyperbaric oxygenation therapy on prognosis and oxidative stress of acute permanent cerebral ischemic stroke in rats

Objective: To evaluate the therapeutic effect and the oxidative stress effect of 9 and 18 hour hyperbaric oxygenation therapy (HBOT) protocols on the earliest stage of acute permanent middle cerebral artery occlusion (MCAO) in rats.Methods: The permanent MCAO model of rats was used. The animals were randomly divided into 9 and 18 hour HBOT groups, as well as a control group.Main outcome measures: (1) The Garcia neurological grading system was used to assess the therapeutic effect of hyperbaric oxygenation therapy; (2) the infarct volume was calculated with the 2,3,5-triphenyltetrazolium chloride (TTC) pathologic staining and NIH Image J software 24 and 120 hours after MCAO; (3) the level of reactive oxygen species determined by superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) in ischemic brain tissue were separately examined at the 18, 48 and 120 hour post-ischemia time points using spectrophotometry.Results: (1) There were significant improvements in the neurobehavioral outcome of the rats in the 9 and the 18 hour groups, as compared with rats from the control group (p<0.01); (2) cerebral infarct volume decreased 63–64% in the rats of 9 hour group and 51–66% in the 18 hour group at the 24 and 120 hour time points, as compared with that of the control group; (3) the SOD levels of the 9 and 18 hour groups were remarkably lower than those of control group after both 18 and 48 hours (p<0.01 and p<0.05); (4) the MDA level of the 9 and 18 hour groups were both remarkably lower than the control groups, especially at 18 hours (p<0.05). Meanwhile, the MDA level in the 9 hour group was remarkably lower than both the 18 hour group and the control group (p<0.01 and p<0.05); (5) the level of NO in both hyperbaric oxygenation therapy groups were remarkably higher than that of the control at 18 and 48 hour time points (p<0.01). While the level in 18 hour group was remarkably lower than that of 9 hour group at 18 hour time point (p<0.05). At the 120 hour mark, the NO levels were basically the same in all the three groups.Conclusions: (1) The two protocols of large dose hyperbaric oxygenation therapy are highly efficient in reducing infarct volume and improving neurobehavioral outcome in permanent MCAO rats within the earliest stages of stroke; (2) increased duration of hyperbaric oxygenation therapy does not appear to equate to improved outcomes; in fact, the longer duration may aggravate the oxidative stress in ischemic tissue.

Effect of Acetyl‐L‐Carnitine on Infarct Size in Multiple Models of Experimental Focal Cerebral Ischemia in Rats

Acetyl‐L‐carnitine (ALCAR), an acetyl ester of carnitine present in brain, is involved in many aspects of neuronal activity, e.g., energy metabolism, phospholipid synthesis for membrane formation/integrity, and modulation of neurotrophic factors and neurohormones. Aberrant neuronal activity occurs following stroke, and ALCAR has been shown to provide some neuroprotection in a few rat studies. This study determined the neuroprotective effect of ALCAR in multiple rat models of ischemic stroke. Focal cerebral ischemia was induced using distal permanent middle cerebral artery occlusion (MCAO) combined with bilateral carotid artery occlusion (BCCO), proximal permanent MCAO, and both permanent and transient intraluminal suture models. Rats were subjected to distal permanent MCAO combined with BCCO and treated with acute or chronic doses of ALCAR 30 min prior the MCAO. Acute doses of 50, 200, 400 mg/kg I.P. did no affect infarct size, while chronic ALCAR (400mg/kg X 5 days) significantly reduced in infarct size (p&lt; 0.001). Lower doses ALCAR (50 and 200 mg/kg X 5 days) failed to alter infarct size. The effective dose of ALCAR (400 X 5 days) also produced a significant reduction in infarct size in rats subjected to proximal permanent MCAO, permanent, and transient intraluminal suture models. ALCAR might exert its neuroprotection via a host of different mechanistic pathways.

Modified permanent middle cerebral artery occlusion rat model aiming to reduce variability in infarct size

In animal cerebral infarct experiments, the most important aspect is to produce consistent infarct size and localization. In an attempt to improve the conventional middle cerebral artery (MCA) coagulation technique, we developed a new animal model using a microclip to reduce variability in infarct size. Male Sprague–Dawley rats were subjected to right MCA occlusion. The animals were divided into two groups; conventional MCA occlusion group (Group 1; n = 9) and modified clip occlusion group (Group 2; n = 9). In Group 2, the proximal portion of MCA was occluded by applying a small clip just proximal to the olfactory nerve, and the MCA from the clipped position to the position just proximal to the level of the inferior cerebral vein was electrocoagulated using a bipolar diathermy in the same manner as in Group 1. In other words, the only difference between these two groups was the manner of occlusion of the most proximal portion of the MCA. Rats were killed 24 hours after the stroke-inducing surgery, and infarct volume was determined by an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. The cortical infarct volumes were 51.0 ± 13.8% in Group 1 and 46.3 ± 6.2% in Group 2. The scattering of cortical infarct volume was significantly small in Group 2 (p=0.0176). The differences in scattering of striatal and total infarct volumes did not reach statistical significance. The present results demonstrated that the new MCA occlusion model using a clip significantly reduces the variability in cortical infarct volume, solving the problems of the model using coagulation alone. That permanent MCA occlusion model using a clip is an excellent method that produces more consistent and reproducible infarction.

NXY-059

Marc Fisher MD Kennedy Lees MD Section Editors The SAINT I trial published in the February 9, 2006 issue of the New England Journal of Medicine represents the first “positive” neuroprotective trial in stroke.1 This breaks a long string of “neuroprotective” failures and indicates some hope for stroke treatment.2 Is NXY-059 a better drug than previous failed agents or was the trial just better designed? Probably both. NXY-059 is the first agent that fulfilled all the Stroke Therapy Academic Industry Roundtable (STAIR) recommendations regarding preclinical development before it entered a large phase-III clinical trial in stroke.3 The preclinical data with NXY-059 were robust and impressive. NXY-059 was effective at both reducing infarct size and improving functional outcome in both temporary and permanent rodent middle cerebral artery (MCA) occlusion models.4,5 There was a clear dose response and the therapeutic window extended out to 4 hours.5 Next, NXY-059 was tested in a nonhuman primate model, albeit the lissencephalic marmoset.6,7 Again, NXY-059 both reduced infarct size and improved long-term (10-week) functional outcome (reduced neglect and improved arm function) with a 4-hour window in a permanent MCA occlusion model.7 The reduction in infarct size and functional improvement was better than that observed with other neuroprotective agents (the GABA mimetic agent, clomethiazole and the NMDA antagonist, AR-R15896AR) …

The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia

A novel fluorescence probe, 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) was used to investigate the generation of highly reactive oxygen species (hROS) under ischemia both in vitro and in vivo. In the in vitro study, HT 22 cells were used to demonstrate that was predominantly detected in the cytoplasm, which coincides with the location of the mitochondria and then its HPF fluorescence gradually increased from 6 to 24 h due to glutamate induced oxidative stress. In the in vivo study, the permanent and transient middle cerebral artery occlusion (MCAO) was induced in rats. Brain slices were incubated in an artificial medium containing HPF. The area of enhanced HPF fluorescence existed in both the ischemic core and the peri-infarct area at 4 h after MCAO in both permanent and transient MCAO models. The area extended beyond the boundary of the ischemic damage into biochemically viable tissue. The enhanced fluorescent intensity following transient MCAO was higher than that observed in the permanent MCAO model. Hydroxyl radical scavenger, MCI-186 significantly suppressed the enhanced fluorescence intensity. This study demonstrated that HPF has a high sensitivity and specificity for the detection of hROS in focal cerebral ischemia as well as in a cellular model of oxidative stress.