Suture Middle Cerebral Artery Occlusion Research Articles

Abstract WP106: Optogenetic Neuronal Stimulation Reduces Neuronal Nitric Oxide Synthase After Stroke

Objective: Nitric oxide (NO) is a gaseous messenger that mediates many physiological functions, including neuronal excitability, vascular tone and angiogenesis. Nitric oxide synthases are a family of enzymes that catalyzes the production of NO. While activation of endothelial NOS (eNOS) has been shown to be neuroprotective, activation of neuronal NOS (nNOS) and inducible NOS (iNOS) worsens ischemic damage. Although the role of NOS in acute stroke is well-established, their contribution in stroke recovery is less clear. Our lab recently demonstrated that optogenetic neuronal stimulation can enhance post-stroke recovery. As NOS mediates many physiological processes important for stroke recovery, we investigate whether NOS is involved in stimulation-enhanced recovery. We hypothesize that neuronal stimulations promote recovery by altering NOS expression. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent experimental stroke induced by an intraluminal middle cerebral artery suture occlusion. Repeated optogenetic stimulations in the contralesional cerebellar dentate nucleus (cLCN) were administered from post-stroke day5-14. Functional recovery was evaluated by sensory-motor behavior tests. Mice were sacrificed at post-stroke day15, ipsilesional and contralesional motor and sensory cortices were dissected. qPCR was performed to examine expression of nNOS, iNOS and eNOS. Results: Our data showed that stimulated stroke mice recovered quickly, with significantly faster rotating beam speed at day14 post-stroke (p<0.001). Quantitative PCR revealed that stimulations significantly reduced nNOS expression, while no change was detected for eNOS and iNOS. Interestingly, nNOS was significantly reduced in the contralesional primary motor cortex in cLCN-stimulated stroke mice (p<0.001), suggesting the involvement of nNOS in the contralesional cortex in stroke recovery. Conclusion: Optogenetic stimulations promote post-stroke recovery and selectively reduced nNOS expression in the contralesional motor cortex. Current ongoing studies examine the nNOS signaling pathway after stimulation and the effect of manipulating nNOS expression in stroke recovery.

Abstract TP259: Surrogate MRI Signatures for Contrast Enhanced Imaging That Predict Acute Blood-brain Barrier Damage in Ischemia-reperfusion

Background and Purpose: Contrast-enhanced magnetic resonance imaging (CE-MRI) is used to measure blood-brain barrier (BBB) damage after stroke, but is contra-indicated in patients with compromised kidney function. Thus, there is a need for non-contrast MRI techniques that portend BBB damage. We examined the relationships between CE-MRI and non-contrast MRI measures to test whether the latter serve as surrogate biomarkers of BBB damage in acute stroke. Methods: Male Wistar rats (∼300 g; N=22) were subjected to focal cerebral ischemia-reperfusion using a middle cerebral artery suture occlusion model. They were imaged in a 7 Tesla Bruker MRI system. The parameters measured were: cerebral blood flow (CBF), T 2 , T 1 and T 1 -under-off resonance-saturation (T 1sat ). All data except CBF were expressed as ipsilateral-to-contralateral ratios (I-C). Post-reperfusion BBB damage was measured by CE-MRI via blood-to-brain forward volumetric transfer constant (K trans ) maps. CBF, T 2 , T 1 and T 1sat were compared to K trans from the same region of interest (ROI). Scatterplots with Pearson correlation coefficients (r) were used to compare the data and significances inferred at p <0.05. Results: Preoptic area (PoA) and striatum (Str) were found ischemic in nearly all rats, with neocortical areas lesser affected. During occlusion, CBF in PoA and Str were 26±15 and 42±18 ml/100g/min (20-25% of contralateral side), respectively. After reperfusion, CBF was 78±27 and 99±50 ml/100g/min in PoA and Str, respectively. Contrast enhancement or BBB damage was observed in the PoA in all 22 rats and in 17 in Str. The I-C values of T 2 , T 1 and T 1sat elevated between occlusion and reperfusion periods and were associated with increased K trans values ( p =0.05). The extent of CBF reduction during occlusion was significant and correlated inversely with increased K trans (r=-0.5; p =0.03), but this relationship was lost after reperfusion (r=0.3; p =0.3). Conclusions: The data suggest that reduction in CBF during occlusion and post-reperfusion elevation of T 2 , T 1 , and T 1sat (i.e. vasogenic edema) are reliable predictors of impending BBB damage in acute stroke. With further confirmation, non-contrast based MRI evaluation of the BBB in acute stroke may be utilized in cases where CE-MRI is not possible.

Acute Hyperglycemia Does Not Affect Brain Swelling or Infarction Volume After Middle Cerebral Artery Occlusion in Rats.

Stroke disproportionally affects diabetic and hyperglycemic patients with increased incidence and is associated with higher morbidity and mortality due to brain swelling. In this study, the intraluminal suture middle cerebral artery occlusion (MCAO) model was used to examine the effects of blood glucose on brain swelling and infarct volume in acutely hyperglycemic rats and normo-glycemic controls. Fifty-four rats were distributed into normo-glycemic sham surgery, hyperglycemic sham surgery, normo-glycemic MCAO, and hyperglycemic MCAO. To induce hyperglycemia, 15 min before MCAO surgery, animals were injected with 50 % dextrose. Animals were subjected to 90 min of MCAO and sacrificed 24 h after reperfusion for hemispheric brain swelling and infarct volume calculations using standard equations. While normo-glycemic and hyperglycemic animals after MCAO presented with significantly higher brain swelling and larger infarcts than their respective controls, no statistical difference was observed for either brain swelling or infarct volume between normo-glycemic shams and hyperglycemic shams or normo-glycemic MCAO animals and hyperglycemic MCAO animals. The findings of this study suggest that blood glucose does not have any significant effect on hemispheric brain swelling or infarct volume after MCAO in rats.

Abstract T MP19: Optogenetic Stimulation of Cerebellar Dentate Nucleus Promotes Persistent Functional Recovery After Stroke

Objective: Functional recovery after stroke has been observed in both human and animal studies. Post-stroke brain stimulations are promising neurorestorative techniques as they allow direct manipulation of the target area’s excitability. Previously we have demonstrated that optogenetic neuronal stimulation of the ipsilesional primary motor cortex promotes functional recovery. To determine an optimal brain stimulation target, we test whether optogenetic neuronal stimulation of the contralesional cerebellar dentate nucleus (cLCN) can promote recovery. We hypothesize that stimulation of cLCN may be more effective, as it sends excitatory outputs to multiple motor and premotor areas. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in cLCN, followed by an intraluminal middle cerebral artery suture occlusion. Three groups of mice were used: control non-stimulated stroke mice, short stim stimulated stroke mice (day5-14 post-stroke) and long stim stimulated stroke mice (day5-28 post-stroke). Sensorimotor behavior tests (rotating beam tests) were used to assess their recovery at day 0, 4, 7, 10, 14, 21 and 28 post-stroke. Results: Our data showed that stimulated stroke mice recovered quickly, with significant improvement in distance traveled as early as day7 (p<0.05), and faster speed at day14 post-stroke (p<0.001). To evaluate whether the effect of cLCN stimulation was persistent, we tested the effects of short stim (day5-14) and long stim (day5-28) on recovery. Interestingly, the short stim group continued to recover after day14 without further stimulations and the long stim group did not further enhance recovery, indicating that functional outcome of cLCN stimulation is persistent, and prolonged stimulations may not be necessary to achieve permanent recovery. Analysis of pCREB activation showed that cLCN stimulation activates the dentatothalamocortical pathway. Conclusion: Our data suggest that cLCN stimulations post-stroke can promote functional recovery, and this pro-recovery effect is persistent. Current studies examine the mechanisms of cLCN-induced recovery, including cortical excitability and synaptic/plasticity markers.

Collateral circulation prevents masticatory muscle impairment in rat middle cerebral artery occlusion model.

The rat suture middle cerebral artery occlusion (MCAO) is a frequently used animal model for investigating the mechanisms of ischemic brain injury. During suture MCAO, transection of the external carotid artery (ECA) potentially restrains blood flow and impairs masticatory muscle and other ECA-supported territories, consequently influencing post-operation animal survival. This study was aimed at investigating the effect of ECA transection on the hemodynamic alterations using a novel synchrotron radiation (SR) angiography technique and magnetic resonance imaging in live animals. Fifteen male adult Sprague-Dawley rats were used in this study. Animals underwent MCAO, in which the ECA was transected. SR angiography was performed before and after MCAO. Rats then underwent magnetic resonance imaging (MRI) to detect the tissue lesion both intra- and extra-cranially. Animals with SR angiography without other manipulations were used as control. High-resolution cerebrovascular morphology was analyzed using a novel technique of SR angiography. The masticatory muscle lesion was further examined by hematoxylin and eosin staining. MRI and histological results showed that there was no masticatory muscle lesion at 1, 7 and 28 days following MCAO with ECA transection. In normal condition, the ECA and its branch external maxillary artery were clearly detected. Following ECA transection, the external maxillary artery was still observed and the blood supply appeared from the anastomotic branch from the pterygopalatine artery. SR angiography further revealed the inter-relationship of hemisphere extra- and intra-cranial vasculature in the rat following MCAO. Transection of the ECA did not impair masticatory muscles in rat suture MCAO. Interrupted blood flow could be compensated by the collateral circulation from the pterygopalatine artery.

Embolic middle cerebral artery occlusion (MCAO) for ischemic stroke with homologous blood clots in rats.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.(1) in 1992 and further characterized by Zhang et al. in 1997(2). Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Abstract W P96: Optogenetic Neuronal Stimulation Enhances Neurotrophin Expression in the Contralesional Motor Cortex After Stroke

Objective: Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity. One form of cortical reorganization involves the balance of interhemispheric interactions between ipsilesional and contralesional cortex. Stimulation of ipsilesional primary motor cortex (iM1) has been shown to be beneficial, however, the role of the contralesional M1 (cM1) remains controversial. Recently we showed that optogenetic stimulations of iM1 post-stroke promote functional recovery. In this study, we investigate the role of contralesional cortex in recovery by optogenetically stimulating iM1 or cM1 and examine the involvement of activity-dependent neurotrophins. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in either iM1 or cM1, followed by an intraluminal middle cerebral artery suture occlusion. Optogenetic stimulation began at day5 post-stroke and continued until day14 post-stroke. Sensorimotor behavior tests were used to assess their recovery at day 0, 2, 7, 10 and 14 post-stroke. Mice were sacrificed at day15 post-stroke and neurotrophin expressions were examined using quantitative PCR. Results: Repeated iM1 stimulations promoted functional recovery at day14 post-stroke, with improved motor performance on the rotating beam test (p<0.01). Real-time PCR revealed significant increases of neurotrophin expressions in contralesional M1 at day15 post-stroke, including brain-derived neurotrophic factor (BDNF) (p<0.05), nerve growth factor (NGF) (p<0.05) and neurotrophin 3 (NTF3) (p<0.05). BDNF and NTF3 expression were also significantly increased in the contralesional S1 of stimulated mice (p<0.05). Conclusion: Our data suggest that activity-dependent neurotrophins in the contralesional cortex may be an important mechanism mediating stroke recovery. Current studies include specific stimulation and inhibition of the iM1 or cM1 post-stroke to elucidate the neurocircuitry mediating stroke recovery. In addition, the expression of neurotrophins will be examined in these studies to elucidate their role in the recovery process.

Abstract T P212: The Impact of the Method of Ischemia/reperfusion Injury on Stroke Outcomes in Type 2 Diabetes

Diabetes worsens functional outcome after ischemic stroke and is associated with greater hemorrhagic transformation (HT) occurrence. We have shown that male diabetic Goto-Kakizaki (GK) rats develop greater HT and neurological deficit despite smaller infarcts after temporary (3/21 hr) middle cerebral artery occlusion (MCAO) induced by the suture model. The impact of the duration of ischemia/reperfusion (I/R) and the method of ischemia on neurovascular injury and functional outcome in diabetic stroke remain unknown. To address this gap, control Wistar and diabetic GK rats were subjected to 90 min/23 hr, 3 hr/21 hr, or 3 hr/7 day MCAO by suture occlusion or embolus placement. In all groups, infarct size, edema, HT occurrence and severity (hemoglobin level), and functional outcome (grip strength and composite deficit score) were measured (Table). Infarct size was smaller in GK rats with suture or embolic MCAO, but expanded with longer reperfusion period. Edema, HT occurrence and severity were all increased in GK rats after 90 min and 3 hr occlusion with the suture model, but not in the embolic MCAO. Motor deficit was greater in diabetic rats. These findings suggest that diabetes accelerates the development of HT and amplifies vascular damage especially in the suture model where blood flow is rapidly reestablished. These results highlight the importance of earlier vascular protection to improve neurological outcome of acute ischemic stroke in diabetes.

Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model

Intraluminal suture middle cerebral artery occlusion (MCAO) model is the most frequently used model for ischemic stroke. However, the success rate of this model is variable among different research studies. This study aimed to investigate the effect of postischemic temperature on the success rate. A total of 100 C57BL/6 mice were randomized into two groups: control group (n=50), body temperature was allowed to self-regulate after MCAO; temperature-controlled group (n=50), mice were kept warm in an incubator for 12 h after MCAO. The body temperature of animals was measured before, during, and for 12 h after MCAO. Neurological deficits and infarct volumes were measured at 24 h after MCAO. There was significant difference (P<0.05) of the body temperature between the two groups from 0.5 h to 3.5 h post ischemia. Moreover, there was obvious difference between the success rates of the two groups (control group: 52%, temperature-controlled group: 84%, P<0.05). In the successful models, infarct volume was significantly (P<0.05) higher in temperature-controlled group (53.44%±9.83%, n=42) than control group (45.63%±10.24%, n=26). There was significant difference of the modified neurological severity scores (P<0.05), left adhesive tests (P<0.05) between the two groups. Our data demonstrated that postischemic warming contributed to the success of mouse MCAO model.

Comparative analysis of the neurovascular injury and functional outcomes in experimental stroke models in diabetic Goto-Kakizaki rats

Diabetes worsens functional outcome and is associated with greater hemorrhagic transformation (HT) after ischemic stroke. We have shown that diabetic Goto-Kakizaki (GK) rats develop greater HT and neurological deficit despite smaller infarcts after transient middle cerebral artery occlusion (MCAO) with the suture model. However, the impact of (1) the duration of ischemia/reperfusion (I/R); (2) the method of ischemia; and (3) acute glycemic control on neurovascular injury and functional outcome in diabetic stroke remained unanswered. Wistar and GK rats were subjected to variable MCAO by suture or embolus occlusion. A group of GK rats were treated with insulin or metformin before stroke with suture occlusion. In all groups, infarct size, edema, HT occurrence and severity, and functional outcome were measured. Infarct size at 24h was smaller in GK rats with both suture and embolic MCAO, but expanded with longer reperfusion period. Edema and HT were increased in GK rats after 90min and 3h occlusion with the suture model, but not in the embolic MCAO. Neurological deficit was greater in diabetic rats. These findings suggest that diabetes accelerates the development of HT and amplifies vascular damage in the suture model where blood flow is rapidly reestablished. Acute metformin treatment worsened the infarct size, HT, and behavior outcome, whereas insulin treatment showed a protective effect. These results suggest that the impact of ischemia/reperfusion on neurovascular injury and functional outcome especially in disease models needs to be fully characterized using different models of stroke to model the human condition.

Spatiotemporal Uptake Characteristics of [ 18 ]F-2-Fluoro-2-Deoxy- d -Glucose in a Rat Middle Cerebral Artery Occlusion Model

Alterations of cerebral glucose metabolism are well anticipated during cerebral ischemia. However, detailed spatiotemporal characteristics of disturbed cerebral glucose metabolism during acute ischemia remain largely elusive. This study aims to delineate spatiotemporal distributions of [18]F-2-fluoro-2-deoxy-D-glucose (FDG) uptake using positron emission tomography imaging, particularly at the peri-ischemic zone, and its correlation with tissue outcome. The intraluminal suture middle cerebral artery occlusion model was used to induce focal cerebral ischemia in rats (n=48). All animals underwent sequential MRI and FDG positron emission tomography imaging at different times (30-150 minutes) after middle cerebral artery occlusion. MR and positron emission tomography images were coregistered. FDG uptake in the peri-ischemic zone was assessed in relation to middle cerebral artery occlusion duration, cerebral blood flow, apparent diffusion coefficient, and 24-hour T2 lesions. Elevated FDG uptake was consistently observed at the peri-ischemic zone surrounding the presumed ischemic core with low FDG uptake. Both the spatial volume and the uptake level of the hyper-uptake region were inversely correlated with the duration of middle cerebral artery occlusion. The hyper-uptake regions exhibited a mild reduction of cerebral blood flow (28.2±3.2%) and apparent diffusion coefficient (9.1±1.4%) when compared with that in the contralateral hemisphere. Colocalization analysis revealed that, with reperfusion, an average of 12.1±1.7% of the hyper-uptake volume was recruited into final infarction. Elevated FDG uptake at the peri-ischemic zone is consistently observed during acute cerebral ischemia. The region with elevated FDG uptake likely reflects viable tissues that can be salvaged with reperfusion. Therefore, acute FDG positron emission tomography imaging might hold promise in the management of patients with acute stroke.

Role of suture diameter and vessel insertion position in the establishment of the middle cerebral artery occlusion rat model

The aim of the present study was to explore the role of suture diameter and vessel insertion position in the preparation of the middle cerebral artery occlusion (MCAO) rat model. A total of 84 Sprague-Dawley rats (weighing 250–300 g) were randomly divided to three groups: group A (type 1.0, suture diameter 0.16–0.17 mm and tip 0.21–0.22 mm); group B (type 2.0; suture diameter, 0.22–0.23 mm; tip, 0.27–0.28 mm); and group C (type 3.0; suture diameter, 0.28–0.29 mm; and tip, 0.33–0.34 mm). The animals in each group were then subdivided into two subgroups, one of which received a nylon line inserted through the external carotid artery (ECA insertion), while the other received the nylon line through the common carotid artery (CCA insertion) subsequent to a middle or lateral neck incision. The neurological deficit score was evaluated at 4, 8, 24, 48 and 72 h post-surgery. The ischemic brain tissue was stained by 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of the infarct volume. The cerebral edema rate, cerebral infarction volume rate, relative standard deviation (RSD) of the cerebral infarction rate and the success rate were also assessed. The rectal temperature, PaO2, PaCO2, pH, blood pressure and blood glucose levels were controlled and did not vary between the group types. The results suggested that suture diameter and insertion route affected the infarct volume and success rate in the establishment of the suture MCAO rat model. Furthermore, the MCAO model with a 0.22–0.23 mm diameter suture and CCA insertion route provided the highest success rate in the SD rats.

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&lt;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&lt;0.05). Additionally, a significantly faster regain of body weight was observed in stimulated mice after stroke (p&lt;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&lt;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.

The protective effects of T cell deficiency against brain injury are ischemic model-dependent in rats

Previous studies have reported that T cell deficiency reduced infarct sizes after transient middle cerebral artery (MCA) suture occlusion in mice. However, how reperfusion and different models affect the detrimental effects of T cells have not been studied. We investigated the effects of T cell deficiency in nude rats using two stroke models and compared their infarct sizes with those in WT rats. In the distal MCA occlusion (MCAo) model, the distal MCA was permanently occluded and the bilateral common carotid arteries (CCAs) were transiently occluded for 60min. In the suture MCAo model, the MCA was transiently occluded for 100min by the insertion of a monofilament suture. Our results showed that T cell deficiency resulted in about a 50% reduction in infarct size in the suture MCAo model, whereas it had no effect in the distal MCAo model, suggesting the protective effects of T cell deficiency are dependent on the ischemic model used. We further found more total T cells, CD4 T cells and CD8 T cells in the ischemic brains of WT rats in the suture MCAo model than in the distal MCAo model. In addition, we detected more CD68-expressing macrophages in the ischemic brains of WT rats than in nude rats in the suture MCAo but not the distal MCAo model. Lymphocyte reconstitution in nude rats resulted in larger infarct sizes in the suture MCAo, but not in the distal MCAo stroke model. The results of regional CBF measurement indicated a total reperfusion in the MCAo model but only a partial reperfusion in the distal MCAo model. In conclusion, the protective effects of T cell deficiency on brain injury are dependent on the ischemic model used; likely associated with different degrees of reperfusion.

CXCR4 Antagonist AMD3100 Protects Blood–Brain Barrier Integrity and Reduces Inflammatory Response After Focal Ischemia in Mice

Inflammatory response plays a critical role in propagating tissue damage after focal cerebral ischemia. CXCL12 is a key chemokine for leukocyte recruitment. However, the role of CXCL12 and its receptor CXCR4 in ischemia-induced inflammatory response is unclear. Here we use the pharmacological antagonist of CXCR4, AMD3100, to investigate the function of CXCL12/CXCR4 in regulating inflammatory response during acute ischemia. Adult male CD-1 mice (n=184) underwent permanent suture middle cerebral artery occlusion (MCAO). AMD3100 was injected for 3 days (1 mg/kg/day) after MCAO. Brain water content, infarct volume, neurological score, and myeloperoxidase (MPO) expression and activity were examined at 24, 48, and 72 hours after MCAO. Proinflammatory cytokine RNA and protein levels in brain tissue were measured by RT-PCR and enzyme linked immunosorbent assay. Neurological score was greatly improved in AMD3100-treated mice compared with the control mice 3 days after MCAO (P<0.05). Brain edema-induced change of water content, IgG protein leakage, Evans blue extravasation, occludin, and ZO-1 expression in ipsilateral hemisphere were alleviated by acute treatment of AMD3100. MPO expression and activity revealed that AMD3100 profoundly reduced the number of MPO-positive cells in the ischemic region (P<0.05). It also attenuated proinflammatory cytokines including interleukin 6, tumor necrosis factor α, and interferon γ; their mRNA and protein levels changed accordingly compared with the controls (P<0.05). CXCR4 antagonist AMD3100 significantly suppressed inflammatory response and reduced blood-brain barrier disruption after MCAO. AMD3100 attenuated ischemia-induced acute inflammation by suppressing leukocyte migration and infiltration, in addition to reducing proinflammatory cytokine expression in the ischemic region.

New Evidence of Neuroprotection by Lactate after Transient Focal Cerebral Ischaemia: Extended Benefit after Intracerebroventricular Injection and Efficacy of Intravenous Administration

Background: Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously. Methods: Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na <smlcap>l</smlcap>-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na <smlcap>l</smlcap>-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h. Results: Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm³, n = 7 vs. 12.1 ± 3.8 mm³, n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm³, n = 12 vs. 29.6 ± 25.4 mm³, n = 12, p < 0.05). Conclusions: The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.

Fastigial Nucleus Electrostimulation Reduces the Expression of Repulsive Guidance Molecule, Improves Axonal Growth Following Focal Cerebral Ischemia

The role of repulsive guidance molecule A (RGMa) in cerebral ischemia remains unclear. In the study, we examined the expression of RGMa in ischemic brain tissues following focal cerebral ischemia/reperfusion (IR) in rats. An established middle cerebral artery suture occlusion model was employed. A dipolar electrode was placed into the cerebellum to stimulate the cerebellar fastigial nucleus for 1h at 2h after ischemia. Reverse transcription-polymerase chain reaction was used to measure the mRNA RGMa and its downstream mediator, Ras homolog A (RhoA). Immunohistochemistry was applied to detect RGMa and RhoA expressions and to evaluate axonal regeneration by optical density analysis of 200kDa neurofilaments. We found that both mRNA and protein levels of RGMa and RhoA were increased in the ischemic cortex and hippocampus 48h following cerebral IR and these elevated levels were maintained for 2weeks. Electrostimulation of the fastigial nucleus reduced the expression of RGMa and RhoA at 24h and 2weeks following cerebral IR. In addition, axonal growth was enhanced in the fastigial nucleus electrostimulated group compared to non-stimulated ischemic animals (P<0.05). RGMa/RhoA expression was negatively correlated with the growth of axons (P<0.05). Therefore, we concluded that RGMa and RhoA could be another key molecule and might inhibit axonal regeneration during cerebral IR injury. Electrostimulation of the fastigial nucleus enhances axonal growth, possibly by reducing the expression of RGMa and RhoA after cerebral IR.

Distinctive Effects of T Cell Subsets in Neuronal Injury Induced by Cocultured Splenocytes In Vitro and by In Vivo Stroke in Mice

T cells and their subsets modulate ischemic brain injury. We studied the effects of the absence of T cell subsets on brain infarction after in vivo stroke and then used an in vitro coculture system of splenocytes and neurons to further identify the roles of T cell subsets in neuronal death. Stroke was induced by middle cerebral artery suture occlusion in mice and infarct sizes were measured 2 days poststroke. Splenocytes were cocultured with neurons, and neuronal survival was measured 3 days later. A deficiency of both T and B cells (severe combined immunodeficiency) and the paucity of CD4 or CD8 T cells equally resulted in smaller infarct sizes as measured 2 days poststroke. Although a functional deficiency of regulatory T cells had no effect, impaired Th1 immunity reduced infarction and impaired Th2 immunity aggravated brain injury, which may be due to an inhibited and enhanced inflammatory response in mice deficient in Th1 and Th2 immunity, respectively. In the in vitro coculture system, wild-type splenocytes resulted in dose-dependent neuronal death. The neurotoxicity of splenocytes from these immunodeficient mice was consistent with their effects on stroke in vivo, except for the mice with the paucity of CD4 or CD8 T cells, which did not alter the ratio of neuronal death. T cell subsets play critical roles in brain injury induced by stroke. The detrimental versus beneficial effects of Th1 cells and Th2 cells both in vivo and in vitro reveal differential therapeutic target strategies for stroke treatment.

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.

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.