Intraluminal Middle Cerebral Artery Occlusion Research Articles

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.

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.

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.

A Critical Re-Examination of the Intraluminal Filament MCAO Model: Impact of External Carotid Artery Transection

The intraluminal filament procedure is the most common model of middle cerebral artery occlusion (MCAO). However, consequences of subtle variations in surgical technique on behavioral outcome measures have not been sufficiently explored, which is the aim of this study. Rats randomly received one of three types of transient MCAO (60min) surgeries. The first transected the external carotid artery (ECA) for filament insertion. The other two inserted the filament in the carotid artery (CA), and after reperfusion, the CA was either ligated or blood flow restored. All animals, including shams and naïve controls, were monitored with behavioral tests for 90days. Lesion size and NeuN + cells in the striatum were comparable among MCAO groups. However, rats with ECA transection were consistently lighter than rats with permanent CA ligation, which were lighter than rats with CA reperfusion. Furthermore, rats with ECA transection exhibited the poorest lick efficiency and the greatest impairments in sensorimotor tasks. This study is the first to systematically evaluate the role of ECA transection on functional and morbidity outcomes. Behavioral impairments attributable to the surgical procedure were observed. This confounds studies and is an important issue that needs to be considered when using the intraluminal filament model.

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.

A global transcriptomic view of the multifaceted role of glutathione peroxidase-1 in cerebral ischemic–reperfusion injury

Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1 −/−) mice at 8 and 24 h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ± 1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1 −/−-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1 −/−-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin–proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.

An Optimized Mouse Model for Transient Ischemic Attack

Transient ischemic attacks (TIAs) are brief neurological deficits ofcerebrovascular origin that are followed by complete clinical recovery. Although a plethora of animal models exist for ischemic stroke, a verified TIA model is lacking. We aimed to optimize such a model in mice, investigating the impact of varying durations (from 2.5 to 20 minutes) of intraluminal middle cerebral artery occlusion (MCAo). Three conditions were required to mimic clinical TIA reliably: 1) an objective demonstration of occlusion and reperfusion (assessed by laser Doppler flowmetry); 2) no permanent neurological deficit (assessed by sensorimotor neurological evaluation); and 3) no lesion at 24 hours after reperfusion (assessed by magnetic resonance imaging [MRI]). We observed high incidences of MRI lesions with MCAo durations of 15 minutes or longer. In contrast, no permanent neurological deficits or MRI lesions were observed in animals with MCAo below or equal to 10 minutes. Middle cerebral artery occlusion of 12.5 minutes rarely induced MRI lesions, but histopathologic evaluation using routine and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining revealed minute ischemic changes even after 2.5-minute MCAo. Abundance of necrotic and apoptotic changes gradually increased with the duration of ischemia. These results indicate that 10 minutes or shorter focal cerebral ischemia proves a suitable mouse TIA model; in addition, they indicate that MRI-negative microscopic ischemic damage may occur with even a few minutes of arterial occlusion.

Protective effects of repetitive transcranial magnetic stimulation in a rat model of transient cerebral ischaemia: a microPET study

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive method to excite neurons in the brain. However, the underlying mechanism of its therapeutic effects in stroke remains unclear. The aim of this study was to investigate the neuroprotective effect of high-frequency rTMS in a rat model of transient cerebral ischaemia using positron emission tomography (PET). Sprague-Dawley rats (n=30) were anaesthetized with chloral hydrate and subjected to 90 min of intraluminal middle cerebral artery occlusion (MCAO) with subsequent reperfusion in three groups: control (n=10), rTMS (n=10), or sham-rTMS groups (n=10). In the rTMS group, rTMS was given 1 h after ischaemia and every 24 h for 7 days after MCAO. In all three groups, small-animal PET (microPET) imaging with (18)F-FDG was used to evaluate brain glucose metabolism. Apoptotic molecules were measured in the infarct margin using immunohistochemical staining. The neurological scores of the rats in the rTMS group were higher than in those of the control group over the whole 7-day observation period. The total, cortical and striatal infarct volumes were significantly less in the rTMS group than in the control group, as measured by 2,3,5-triphenyltetrazolium chloride staining. (18)F-FDG microPET images showed significantly higher standardized uptake values in the cortex and striatum in the rTMS group than in the control group in the affected hemisphere. The number of cells positive for caspase-3 was significantly lower in the rTMS group than in the control group, while the Bcl-2/Bax ratio was significantly higher in the rTMS group than in the control group. rTMS therapy increased glucose metabolism and inhibited apoptosis in the ischaemic hemisphere. (18)F-FDG PET could be used to monitor rTMS therapy in transient cerebral ischaemia in animal studies and in future clinical trials.

Ischemic postconditioning may not influence early brain injury induced by focal cerebral ischemia/reperfusion in rats

BackgroundExperimental studies have shown that ischemic postconditioning can reduce neuronal injury in the setting of cerebral ischemia, but the mechanisms are not yet clearly elucidated. This study was conducted to determine whether ischemic postconditioning can alter expression of heat shock protein 70 and reduce acute phase neuronal injury in rats subjected to transient focal cerebral ischemia/reperfusion.MethodsFocal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion for 60 min in twenty male Sprague-Dawley rats (250-300 g). Rats were randomized into control group and an ischemic postconditioning group (10 rats per group). The animals of control group had no intervention either before or after MCA occlusion. Ischemic postconditioning was elicited by 3 cycles of 30 s reperfusion interspersed by 10 s ischemia immediately after onset of reperfusion. The infarct ratios, brain edema ratios and motor behavior deficits were analyzed 24 hrs after ischemic insult. Caspase-3 reactive cells and cells showing heat shock protein 70 activity were counted in the caudoputamen and frontoparietal cortex.ResultsIschemic postconditiong did not reduce infarct size and brain edema ratios compared to control group. Neurologic scores were not significantly different between groups. The number of caspase-3 reactive cells in the ischemic postconditioning group was not significantly different than the value of the control group in the caudoputamen and frontoparietal cortex. The number of cells showing heat shock protein 70 activity was not significantly different than the control group, as well.ConclusionsThese results suggest that ischemic postconditioning may not influence the early brain damage induced by focal cerebral ischemia in rats.

The effects of gabapentin pretreatment on brain injury induced by focal cerebral ischemia/reperfusion in the rat

BackgroundExperimental studies have shown that gabapentin can reduce neuronal injury in the setting of cerebral ischemia, but the mechanisms have not yet been clearly determined. This study was conducted to determine whether gabapentin pretreatment altered expression levels of heat shock protein 70 and reduced acute phase neuronal injury in rats subjected to transient focal cerebral ischemia/reperfusion.MethodsForty male Sprague-Dawley rats (260-300 g) were randomly assigned to one of four groups (saline-treated, or 0.1, 0.5, or 5 mg/kg gabapentin group). In all animals, focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion for 1 hour. The animals of the gabapentin groups were pretreated with a single intravenous administration of gabapentin 20 minutes before ischemic insults. The infarct volume, brain edema and motor behavior deficits were analyzed 24 hours after ischemic insult. Caspase-3-reactive cells and cells showing Hsp70 activity were counted in the caudoputamen and fronto-parietal cortex.ResultsThe infarction ratio was significantly decreased in the 5 mg/kg gabapentin group (P < 0.05) and brain edema ratios were significantly reduced in the 0.1 mg/kg, 0.5 mg/kg, and 5 mg/kg gabapentin groups 24 hours after ischemia/reperfusion injury (P < 0.05). There were more Hsp70-reactive cells in the 5 mg/kg gabapentin group than in the saline group in both the caudoputamen and fronto-parietal cortex (P < 0.05).ConclusionsThese results indicate that gabapentin may have a neuroprotective effect and can reduce early neuronal injury caused by focal cerebral ischemia/reperfusion; this may be mediated by expression of Hsp70. However, gabapentin pretreatment did not prevent caspase-dependent apoptosis.

Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas, FasL and caspase-3 proteins in rats with cerebral ischemia

To observe the effects of Naomaitong, a compound traditional Chinese herbal medicine, combined with mobilization of bone marrow mesenchymal stem cells (BMSCs) on neuron apoptosis in rats with cerebral ischemia, and to explore the possible mechanism by detecting the expressions of Fas, FasL and caspase-3 proteins. Two hundred and two SD rats were divided into sham-operated group, untreated group, recombinant granulocyte colony-stimulating factor (rG-CSF) group, Naomaitong group and Naomaitong plus rG-CSF group (combination group). Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion using a nylon thread with some modification. Rats in the rG-CSF group and the untreated group were administered with rG-CSF 10 microg/(kg x d) by subcutaneous injection 3 d before and 2 d after the operation respectively, once a day, and rats in the Naomaitong group and the combination group were intragastrically administered Naomaitong before and after the operation until sacrificed. Two, three, seven and fourteen days after operation, count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were determined. General neural function score (GNFS) was evaluated. Neuron apoptosis, expressions of Fas, FasL and caspase-3 in rat's brain were all measured. Count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were high in the untreated group, and reached the peak at 3 d and 7 d respectively. CD34 expression in brain tissue was increased in each treated group, especially in the combination group. GNFS was increased at 3 d and 7 d in the untreated group, 7 d and 14 d in the rG-CSF group and the combination group. Expressions of Fas, FasL and caspase-3 were increased 2, 3 and 7 d after operation, while expression of FasL at 2 d in the rG-CSF group, expressions of Fas, FasL and caspase-3 in the combination group were decreased. Expressions of Fas, FasL and caspase-3 at 7 d and 14 d in the combination group were lower than those in the rG-CSF group. Meanwhile, expressions of Fas, FasL and caspase-3 were decreased in each group at 14 d as compared with those at 3 d. There exists interaction between Naomaitong and BMSC mobilization in the effect of improving nerve function and inhibiting neuron apoptosis in rats after cerebral ischemia. It is implied that Naomaitong combined with BMSC mobilization down-regulates the expressions of Fas and FasL in early phase and then inhibits the apoptosis cascade reaction caused by caspase-3, which causes further inhibition of Fas and FasL expression after cerebral ischemia.

Social isolation alters neuroinflammatory response to stroke

Social isolation has dramatic long-term physiological and psychological consequences; however, the mechanisms by which social isolation influences disease outcome are largely unknown. The purpose of the present study was to investigate the effects of social isolation on neuronal damage, neuroinflammation, and functional outcome after focal cerebral ischemia. Male mice were socially isolated (housed individually) or pair housed with an ovariectomized female before induction of stroke, via transient intraluminal middle cerebral artery occlusion (MCAO), or SHAM surgery. In these experiments, peri-ischemic social isolation decreases poststroke survival rate and exacerbates infarct size and edema development. The social influence on ischemic damage is accompanied by an altered neuroinflammatory response; specifically, central interleukin-6 (IL-6) signaling is down-regulated, whereas peripheral IL-6 is up-regulated, in isolated relative to socially housed mice. In addition, intracerebroventricular injection of an IL-6 neutralizing antibody (10 ng) eliminates social housing differences in measures of ischemic outcome. Taken together, these data suggest that central IL-6 is an important mediator of social influences on stroke outcome.

Effect of Acetyl‐L‐Carnitine (ALCAR) on the Striatal Outflow of Various Neurotransmitters Using Microdialysis and the Role of the GLT‐1/EAA‐2 Glutamate Transporter During Middle Cerebral Artery Occlusion (MCAO) in Rats

We have shown that ALCAR is neuroprotective in rat models of ischemia. This study determined ALCAR's effect on extracellular fluid (ECF) neurotransmitters that are disrupted during ischemia. Using striatal microdialysis we measured ALCAR's effect on MCAO‐induced alterations in ECF dopamine (DA), serotonin (5HT), and their major metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC), 4‐hydroxy‐3‐methoxyphenylacetic acid (HVA), and 5‐hydroxyindolacetic acid (5‐HIAA), as well as glutamate (GLU), GABA and taurine (TAU). ECF GLU was also monitored in the presence of dihydrokainate (DHK), a GLT‐1/EAA‐2 inhibitor before, and during permanent intraluminal MCAO. A marked decrease of DA, 5‐HT and their metabolites was observed during MCAO. The amino acids also greatly increased, likely reflecting massive neuronal cell death. Chronic pretreatment with ALCAR (400mg/kg/5days, i.p) attenuated the decrease in DA, DOPAC, HVA, 5‐HIAA (p&lt;0.05), while the ECF level of 5‐HT was unaltered. ALCAR also attenuated the expected increase in GLU, GABA and TAU (p&lt;0.01). In the presence of DHK, ECF GLU significantly increased in both control and ALCAR‐treated groups. The neuroprotection of ALCAR may partly result from restoration of the balance between excitatory and inhibitory neurotransmitters, especially via a reduction in GLU accumulation by modulation of GLT‐1 transporters.

Mechanistic Insight into Neurotoxicity of Tissue Plasminogen Activator-Induced Thrombolysis Products in a Rat Intraluminal Middle Cerebral Artery Occlusion Model

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) after ischemic stroke is effective. However, rtPA potentiates neuronal damage, and interactions between rtPA and thrombolysis products (TLP) have been reported to play a role in this. In the present study we investigated the mechanisms underlying rtPA- and TLP-induced neurotoxicity. Adult male Sprague-Dawley rats were subjected to 60-min intraluminal middle cerebral artery (MCA) occlusion, and then treated with rtPA (10 mg/kg), TLP, or saline. To evaluate the effects of a free radical scavenger, treatment with edaravone and TLP was evaluated. To investigate the role of red blood cells (RBCs), RBC-depleted TLP was used. Neurological deficit scores, infarct volume, and immuno-histochemical localization of oxidative end products for lipid and DNA (4-hydroxy-2-nonenal [4-HNE] and 8-hydroxy-deoxyguanosine [8-OHdG]) were evaluated. TLP increased the infarct volume, worsened the neurological deficits, and increased accumulations of 4-HNE and 8-OHdG. Edaravone treatment significantly reduced the lesion volume and improved the neurological score. Both infarct volume and accumulation of oxidative products were significantly suppressed when RBC-depleted TLP was used. In this mechanical model of MCA occlusion, rtPA-induced TLP, especially in the presence of RBCs, contributed to neuronal damage by accelerating free radical injury.