Intraluminal Middle Cerebral Artery Research Articles

Alteplase Treatment does not Increase Brain Injury After Mechanical Middle Cerebral Artery Occlusion in the Rat

Recanalization of an occluded vessel with recombinant tissue plasminogen activator is an effective strategy for treating acute ischemic stroke. Recombinant tissue plasminogen activator is administered as alteplase, a formulation containing many excipients including L-arginine, the substrate for nitric oxide production. Most studies fail to compare the effects of alteplase on brain injury to its L-arginine carrier solution. This study aimed to verify the previously reported detrimental effects of alteplase after cerebral ischemia and delineate the contribution of L-arginine. Male Wistar rats, subjected to 90 minutes of intraluminal middle cerebral artery occlusion (MCAO), were administered alteplase, the carrier solution or saline upon reperfusion. Neither alteplase nor the carrier affected cerebral blood flow (CBF) restoration throughout the first 60 minutes of reperfusion. Alteplase treatment was associated with increased mortality after MCAO. Twenty-four hours after MCAO, neurologic function and infarct volume did not differ between rats treated with alteplase, the carrier solution, or saline. Irrespective of treatment group, infarct volume was correlated with CBF during reperfusion, neuroscore, and peri-infarct depolarizations. These results suggest that alteplase treatment, independent of thrombolysis, does not cause increased ischemic injury compared with its appropriate carrier solution, supporting the continued use of alteplase in eligible ischemic stroke patients.

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Effects of memantine and melatonin on signal transduction pathways vascular leakage and brain injury after focal cerebral ischemia in mice

Because of their favorable action profiles in humans, both memantine and melatonin are particularly interesting candidates as neuroprotectants in acute ischemic stroke. Until now, the signaling mechanisms mediating memantine’s neuroprotective actions remained essentially uninvestigated. In addition, we have combined memantine with melatonin, which is a well-known neuroprotective molecule. Herein, we examined the effects of memantine (20mg/kg, i.p.) administered alone or in combination with melatonin (4mg/kg, i.p.) on the activation of signaling transduction pathways, IgG extravasation and ischemic injury in mice submitted to 90min of intraluminal middle cerebral artery occlusion, followed by 24h of reperfusion. In these studies, both agents reduced ischemic injury and the density of DNA-fragmentation. Notably, melatonin/memantine combination reduced ischemic injury further as compared with memantine treatment, which was associated with reduced IgG extravasation, indicating vascular leakage in the brain. Animals receiving memantine exhibited elevated ERK-1/2 and decreased p21 and p38/MAPK activations, while it had no significant effect on phosphorylated Akt and SAPK/JNK1/2 in the ischemic brain. However, melatonin increased the activation of Akt and reduced the activations of ERK-1/2, p21, p38/MAPK and SAPK/JNK1/2 significantly. Synergistic effects of memantine and melatonin were observed in the inactivation of p21, p38/MAPK and SAPK/JNK1/2 pathways. Moreover, memantine reversed the effects of melatonin on the activation of ERK-1/2 pathway. Here, we provide evidence that free radical scavenger melatonin potentiates the effects of memantine on ischemic brain injury via inactivations of p21 and stress kinases p38/MAPK and SAPK/JNK1/2 pathways.

Abstract TMP35: Optogenetic Stimulation Of Motor Cortex Neurons Promotes Recovery After Stroke

Objective: Functional recovery after stroke has been observed in both animal and human studies and is currently attributed to both brain remodeling and plasticity. Brain stimulation techniques such as electrical stimulation and transcranial magnetic stimulation have been used successfully to enhance recovery. However, what mediates this recovery is not well understood. Elucidating the mechanism(s) is difficult because these stimulation techniques non-specifically activate all cell types near the stimulation site. Here we use optogenetic techniques to specifically stimulate layer V pyramidal neurons in the ipsilesional motor cortex at day 5 post-stroke, and investigate the effects on functional recovery as well as underlying mechanisms. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in the ipsilesional M1. All mice were then subjected to an intraluminal middle cerebral artery suture occlusion (30min). Optogenetic stimulation began at day 5 post-stroke and continued until day 14 post-stroke. Sensorimotor behavior tests were used to assess their behavioral recovery at day 0, 2, 7, 10 and 14 post-stroke. Body weights were also measured. Changes in cerebral blood flow were measured at day 14 post-stroke using the Laser Doppler Flowmetry. Results: Rotating beam test revealed that stimulated mice recovered significantly faster than non-stimulated control mice at day 10 and 14 after stroke (p<0.05). Stimulated mice also performed significantly better in the adhesive tape test at day 14, with a shorter tape removal time on the contralesional limb (p<0.05). Additionally, a significantly faster regain of body weight was observed in stimulated mice after stroke (p<0.05-0.01). Cerebral blood flow measurements revealed that stimulated mice exhibited significantly larger increase in cerebral blood flow at day 14 post-stroke (p<0.05-0.01). Conclusion: These data indicate that optogenetic stimulation of motor cortex neurons can promote behavioral recovery in mice after stroke. Current studies examine the mechanisms underlying this recovery, including genes related to neurovascular coupling and neurotrophic factors after stimulation.

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3. Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 (1). Because of the ease of genetic manipulation in mice, these models have also been developed in this species (2-3). Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume (4). Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.

In Vivo Imaging of Brain Ischemia Using an Oxygen-Dependent Degradative Fusion Protein Probe

Within the ischemic penumbra, blood flow is sufficiently reduced that it results in hypoxia severe enough to arrest physiological function. Nevertheless, it has been shown that cells present within this region can be rescued and resuscitated by restoring perfusion and through other protective therapies. Thus, the early detection of the ischemic penumbra can be exploited to improve outcomes after focal ischemia. Hypoxia-inducible factor (HIF)-1 is a transcription factor induced by a reduction in molecular oxygen levels. Although the role of HIF-1 in the ischemic penumbra remains unknown, there is a strong correlation between areas with HIF-1 activity and the ischemic penumbra. We recently developed a near-infrared fluorescently labeled-fusion protein, POH-N, with an oxygen-dependent degradation property identical to the alpha subunit of HIF-1. Here, we conduct in vivo imaging of HIF-active regions using POH-N in ischemic brains after transient focal cerebral ischemia induced using the intraluminal middle cerebral artery occlusion technique in mice. The results demonstrate that POH-N enables the in vivo monitoring and ex vivo detection of HIF-1-active regions after ischemic brain injury and suggest its potential in imaging and drug delivery to HIF-1-active areas in ischemic brains.

Using Pimonidazole Delineated Penumbra To Improve Translational Research on Acute Stroke Neuroprotection (SC02.005)

Objective: To improve the translation of animal model findings on stroke neuroprotection into clinical practice, we used pimonidazole (HypoxyProbe) to delineate the penumbra to study its associated biomarkers. In addition we focused on markers within 6 hours of stroke onset as this time period may represent the best window to protect the penumbra from evolving into irreversible damage of ischemic core. Background Translation of animal stroke research has not been successful. For most of the published reports, the outcome measures have been infarct volume or behavioral measures. Perhaps an outcome reflecting biomarkers of penumbra would be more relevant. In addition, there is increased use of penumbra assessment in clinical settings; we need a better understanding of penumbra. Design/Methods: We used twenty Sprague-Dawley rats ages 5 to 24 months for our study. All animal procedures were approved by the Purdue Animal Care and Use Committee. We used an intraluminal middle cerebral artery occlusion (MCAo) model for stroke induction. Occlusion was maintained without reperfusion for 5 hours. Results: Immunostained formalin-fixed sections with antibodies against adducts of reduced pimonidazole demonstrated the location of penumbra with clear border in each animal. The location, morphology, and size of penumbra of each animal varied significantly even with identical MCAo procedures. This variability is similar to what we have observed in clinical stroke research. Pimonidazole stained mostly cytoplasm of neurons and glia with neuropil staining more towards the boundaries of the penumbra. Within the penumbra, we noted peri-nuclear condensation of hypoxia-inducible factor 1 and increase in methionine sulfoxide expression; with q-PCR, we noted a significant increase in heat-shock protein 70 gene expression but not that of methionine sulfoxide reductase. Conclusions: Pimonidazole delineation of penumbra offers a method to study molecular events within the penumbra precisely and to assess early response to putative therapeutic agents in a more clinically relevant fashion. Supported by: Lutheran Foundation. Disclosure: Dr. Chang has nothing to disclose. Dr. Hong-Goka has nothing to disclose.

Abstract 2984: Optical-resolution Photoacoustic Microscopy: a Novel Imaging Modality Capable of Noninvasively Measuring Brain Oxygen Metabolism at the Microvascular Level during Focal Ischemia

Studying oxygen metabolism during brain ischemia has been limited by imaging modalities that have either low resolution but good tissue penetration, or high resolution requiring invasive preparations (open-skull windows). Here, we report a study of cerebrovascular oxygen metabolism in mouse focal ischemia using a novel minimally invasive, high-resolution technique known as optical-resolution photoacoustic microscopy (OR-PAM). Using OR-PAM, we serially imaged cortical vessels through the intact skull of Swiss Webster mice (n=3) before, during, and up to 72 hrs after a 1-hr intraluminal transient middle cerebral artery occlusion (MCAO). Vessels were imaged at 2 different wavelengths (532 & 563 nm) to quantify oxy-, deoxy-hemoglobin and oxygen saturation (sO 2 , Fig , A-C). Oxygen extraction fraction (OEF) was calculated from venous sO 2 values (assuming a constant arterial sO 2 of 95%). Seventy-two hrs after ischemia, animals were sacrificed and whole brains were stained with TTC to delineate infarct at the cortical surface (D). Arterioles were easily distinguished from venules in baseline images (A, mean arterial sO 2 , 87%; mean venous sO 2 , 76%). During MCAO, mean arteriolar sO 2 values did not change; however, venular sO 2 decreased precipitously (B, E), greater in regions that went on to infarct (45±2%) compared to surrounding regions (52±2%, p<0.001). Three and 7 hrs after reperfusion, venous sO 2 slowly recovered but not back to baseline levels. Twenty-four (C) and 72 hrs after reperfusion, venous sO 2 in regions of infarction increased above baseline (83%), approaching arterial sO 2 values of 90%, while venous sO2 in peri-infarct regions (71%) returned near baseline levels of 76% (E). OEF increased from 18% to 55% in regions of eventual infarction, during MCAO; following reperfusion, OEF progressively dropped below baseline levels (12%). In surrounding surviving tissue, OEF returned close to baseline levels (24%) following reperfusion (F). OR-PAM is capable of measuring oxygen metabolism at high resolution during focal ischemia. Consistent with previous PET studies in human stroke, we found OEF increased during acute ischemia; however, OEF remained elevated (above baseline values) after reperfusion, suggesting that injured tissue may have increased oxygen metabolism. OEF gradually decreased below baseline values in infarcted tissue, but returned to near-baseline values in viable peri-infarct regions.

Abstract 2463: Hydrogen Sulifde, A Novel Gasotransmitter, And Ischemic Stroke: Its Protective Role In Cerebral Ischemic - Reperfusion Injury Model

Backgoround: Hydrogen sulfide (H2S) has recently been highligted as the third gasotransmitter following its two counterparts, nitric oxide and carbon monoxide. Several lines of evidence have demonstrated that H2S is a potent vasodilator and has a protective role in ischeic-reperfusion injury of several organs, including heart, liver and kidney. In the present study, we examined the protective effect of H2S in rodent cerebral ischemia-reperfusion injury model. Methods: Ischemia-reperfusion injury model wsa induced by intraluminal middle cerebral artery occlusion (MCAo) for 120 min followed by reperfusion in Male Sprague Dawley rats. Sodium hydrosulfide (NaHS, 1mg/kg and 5mg/kg), the donor of H2S, and normal saline (1mL) was infused via tail vein, immediately after reperfusion. To confirm the protective effect of H2S, we used propargylglycine (PAG)(50mg/kg, intraperitoneal for 7 days befor MCAo). We used Nissl stain to measure infarct sizes, ELISA to determine the levels of IL-1b, IL-6 and TNF-a in the brain, and Western blot to assess the expression of ERK 1/2, STAT 3, phosphorylated STAT3, and Nrf 2. In addition, GSH/GSSG ratio and lipid peroxidase were measured to assess the levels of oxidative stress. Results: Injection of H2S significantly reduced infarct size (5mg/kg: 43 ± 10%; 1mg/kg: 48 ± 12% vs. Control: 58 ± 11%, p < 0.05, n= 9-11). Furthermore, intraperitoneal injection of PAG worsened the infarct size (n=5, p<0.01). Administration of NaHS (5mg/Kg) significantly increased STAT3 phosphorylation at 24 hour after ischemia (n=4, p<0.01). Furthermore, Nrf2, an important molecular in anti-oxidative response, expression signicantly increased in experiment group at 24 and 48 horus after ischemic compared with control (n=4, p<0.01). ELISA showed that the levels of IL-6 increased in experiement gorup (2.5 ± 0.5 ng/mL, n = 4) at 24 and 48 hours after ischemia compared with control gorup (1.9 ± 0.2 ng/mL, n = 4). The levels of IL-1b increased in H2S gorup (0.7 ± 0.1 ng/mL, n = 4) at 48 hours after ischemia but not at 24 hours compred with control (0.4 ± 0.1mg/mL, n = 4). However, TNF-a levels were not different. The levels of GSH/GSSG ratio and lipid perixidase were significantly lower in H2S treated gorup compared with control (p < 0.05, n = 4). Conclusion: In the present study, we showed that administration of NaHS, the donor of H2S, protects brain from ischemia-reperfusion injury. This effect may be driven from anti-oxidative and anti-inflammatory effects of H2S.

Does Acute Behavioral Testing Reflect Successful Ischemia in Rats with Transient Middle Cerebral Artery Occlusion?

Models of intraluminal middle cerebral artery occlusion present an intrinsic variability in infarct size. Behavioral evaluation is frequently performed during arterial occlusion to confirm success of surgery. We compared the value of behavioral testing and multimodal magnetic resonance imaging performed during arterial occlusion for identifying successfully operated animals. Rats were tested with behavioral assessment (using three scoring scales and the adhesive removal test) and multimodal magnetic resonance imaging (including magnetic resonance angiography, diffusion-weighted and perfusion-weighted imaging), both performed during the two-hours of middle cerebral artery occlusion using the intraluminal suture model. Behavioral assessment was repeated 24 h after reperfusion, followed by sacrifice. Acute apparent diffusion coefficient lesion volume was correlated with both 2,3,5-triphenyl tetrazolium chloride infarct size (r = 0·75, P = 0·02) and behavioral status (r = 0·66, P = 0·05) on day one. Conversely, no correlation was found between acute behavioral examination and day one outcomes (2,3,5-triphenyl tetrazolium chloride infarct volume, r = 0·40, P = 0·28; behavioral examination, r = 0·39, P = 0·30). Day zero apparent diffusion coefficient volumes (P = 0·04), but not behavioral assessment (P = 0·60), discriminated animals with day one corticostriatal infarcts from these with subcortical infarcts. Acute behavioral testing performed during arterial occlusion fails to identify successfully operated animals. Acute diffusion magnetic resonance imaging may be more appropriate to assess and reduce infarct size variability in this model.

Liver X Receptor Activation Enhances Blood–Brain Barrier Integrity in the Ischemic Brain and Increases the Abundance of ATP‐Binding Cassette Transporters ABCB1 and ABCC1 on Brain Capillary Cells

The blood-brain barrier (BBB) consists of dense contacts between endothelial cells, the tight junctions, which are complemented by membrane-bound transporters belonging to the ATP-binding cassette (ABC) transporter family. Liver X receptors (LXR) have previously been shown to stabilize the integrity of atherosclerotic noncerebral arteries. Their effects on ischemic cerebral vessels are still unknown. By delivering LXR agonists, T0901317 and GW3965, to mice submitted to 30 minutes intraluminal middle cerebral artery occlusion, we show that LXR activation reduces brain swelling and decreases BBB permeability by upregulating LXR's target calpastatin that deactivates calpain-1/2, stabilizing p120 catenin. p120 catenin specifically interacts with RhoA and Cdc42, inactivating the former and overactivating the latter, thus restoring the postischemic expression, phosphorylation and interaction of the tight junction proteins occludin and zona occludens-1. Moreover, LXR activation deactivates matrix metalloproteases-2/9 and inhibits microvascular apoptosis by deactivating JNK1/2 and caspase-3. In addition to the cholesterol transporters ABCA1 and ABCG1, which have previously been shown to be upregulated by LXR in noncerebral vessels, LXR activation increases the abundance of the drug transporters ABCB1 and ABCC1 on ischemic brain capillaries, as we further show. That LXR activation promotes endothelial integrity in different ways makes this receptor attractive as target for stroke therapies.

Stachybotrys microspora triprenyl phenol-7, a novel fibrinolytic agent, suppresses superoxide production, matrix metalloproteinase-9 expression, and thereby attenuates ischemia/reperfusion injury in rat brain

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a novel fibrinolytic agent with anti-inflammatory effect. Previous study demonstrated that SMTP-7 further ameliorated infarction volume in a mouse embolic stroke model compared with tissue type plasminogen activator (tPA), but the reason SMTP-7 has more beneficial effect than tPA has not yet been determined. In the present study, we investigated whether SMTP-7 has an intrinsic neuroprotective effect against transient focal cerebral ischemia (tFCI). Sprague–Dawley rats were subjected to tFCI by intraluminal middle cerebral artery occlusion for 2 h. Following induction of tFCI, rats were randomized into two groups based on the agent administered: SMTP-7 group and vehicle group. We examined cerebral infarction volume 24 h after reperfusion, and evaluated superoxide production, the expressions of nitrotyrosine and matrix metalloproteinase-9 (MMP-9), which play major roles in secondary brain injury and hemorrhagic transformation. The findings showed that SMTP-7 significantly suppressed superoxide production, the expression of nitrotyrosine and MMP-9 after tFCI, and consequently attenuated ischemic neuronal damage. These results suggest that SMTP-7 has an intrinsic neuroprotective effect on ischemia/reperfusion injury through the suppression of oxidative stress and MMP-9 activation.

Electrical stimulation of olfactory bulb downregulates RGMa expression after ischemia/reperfusion injury in rats

Repulsive guidance molecule A (RGMa) is associated with limited axonal growth after cerebral ischemia. This study focused on the effects of electrical stimulation of olfactory bulb (OB) on the expression of RGMa and axonal regeneration after focal cerebral ischemia/reperfusion injury. Sprague-Dawley (SD) rats were randomly divided into sham-operated group, ischemia/reperfusion (I/R) group (48h, 1 w), stimulation group (48h, 1 w), and sham-stimulated group (48h, 1 w). Focal cerebral ischemia/reperfusion was induced by intraluminal middle cerebral artery occlusion. Electrical stimulation was performed via a bipolar electrode implanted in the right OB. The changes in the expression of RGMa were analyzed by reverse transcription polymerase chain reaction (RT-PCR), Western-blot and immunohistochemistry, respectively. Neurofilament protein 200 (NF-200) immunohistochemical staining was used to assess axonal regeneration. Another group of rats were divided into sham-operated group, I/R group, sham-stimulated group and stimulation group. The behavioral test was conducted using the modified neurological severity score (mNSS). The infarct volume was determined by triphenyltetrazolium chloride (TTC) staining. The levels of RGMa were significantly elevated after ischemia/reperfusion injury. Stimulation treatment downregulated the expression of RGMa, reduced infarct volume and improved neurological function. These observations demonstrated that electrical stimulation of OB might promote axonal regeneration and function recovery after ischemic cerebral injury.

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion in the United States in 2009. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi in 1986 to simulate this impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa. Both techniques have been widely used to identify molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities. This relatively noninvasive method in rats has been extended to use in mice to take advantage of transgenic and knockout strains. To model focal cerebral ischemia, an intraluminal suture is advanced via the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction.

Brain-Targeting Form of Docosahexaenoic Acid for Experimental Stroke Treatment: MRI Evaluation and Anti-Oxidant Impact

Epidemiologic studies report cardiovascular protection conferred by omega-3 fatty acids, in particular docosahexaenoic acid (DHA). However, few experimental studies have addressed its potential in acute stroke treatment. The present study used multimodal MRI to assess in vivo the neuroprotection conferred by DHA and by a brain-targeting form of DHA-containing lysophosphatidylcholine (AceDoPC) in experimental stroke. Rats underwent intraluminal middle cerebral artery occlusion (MCAO) and were treated at reperfusion by intravenous injection of i) saline, ii) plasma from donor rats, iii) DHA or iv) AceDoPC, both solubilized in plasma. Twenty-four hours after reperfusion, animals underwent behavioral tests and were sacrificed. Multiparametric MRI (MRA, DWI, PWI, T2-WI) was performed at H0, during occlusion, and at H24, before sacrifice. Brain tissue was used for assay of F(2)-isoprostanes as lipid peroxidation markers. Initial lesion size and PWI/DWI mismatch were comparable in the four groups. Between H0 and H24, lesion size increased in the saline group (mean ± s.d.: +18% ± 20%), was stable in the plasma group (-3% ± 29%), and decreased in the DHA (-17% ± 15%, P=0.001 compared to saline) and AceDoPC (-34% ± 27%, P=0.001 compared to saline) groups. Neuroscores in the AceDoPC group tended to be lower than in the other groups (P=0.07). Treatments (pooled DHA and AceDoPC groups) significantly decreased lipid peroxidation as compared to controls (pooled saline and vehicle) (P=0.03). MRI-based assessment demonstrated the neuroprotective effect of DHA in the MCAO model. Results further highlighted the therapeutic potential of engineered brain-targeting forms of omega-3 fatty acids for acute stroke treatment.

Positron emission tomography with 11C-flumazenil in the rat shows preservation of binding sites during the acute phase after 2h-transient focal ischemia

Background and purpose: Positron emission tomography (PET) studies in humans have used 11C-flumazenil (FMZ) to assess neuronal viability after stroke. Here we aimed to study whether 11C-FMZ binding was sensitive to neuronal damage in the acute phase following ischemia/reperfusion in the rat brain. Experimental procedures: Transient (2 h followed by reperfusion) and permanent intraluminal middle cerebral artery occlusion was carried out. 11C-FMZ binding was studied by PET up to 24 h after the onset of ischemia. Tissue infarction was evaluated post-mortem at 24 h. Immunohistochemistry against a neuronal nuclei specific protein (NeuN) was performed to assess neuronal injury. Results: No decrease in 11C-FMZ binding was detected in the ipsilateral cortex up to 24 h post-ischemia in the model of transient occlusion despite the fact that rats developed cortical and striatal infarction, and neuronal injury was clearly apparent at this time. In contrast, 11C-FMZ binding was significantly depressed in the ipsilateral cortex at 24 h following permanent ischemia. Conclusions: This finding evidences that 11C-FMZ binding is not sensitive to neuronal damage on the acute phase of ischemia/reperfusion in the rat brain.