A surgical model of permanent and transient middle cerebral artery stroke in the sheep

Background

Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion.

Materials and Methods

18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO.

Results

Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits.

Conclusions

Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.     

Bringing the hospital to the patient: first treatment of stroke patients at the emergency site

Background

Early treatment with rt-PA is critical for favorable outcome of acute stroke. However, only a very small proportion of stroke patients receive this treatment, as most arrive at hospital too late to be eligible for rt-PA therapy.

Methods and Findings

We developed a “Mobile Stroke Unit”, consisting of an ambulance equipped with computed tomography, a point-of-care laboratory system for complete stroke laboratory work-up, and telemedicine capabilities for contact with hospital experts, to achieve delivery of etiology-specific and guideline-adherent stroke treatment at the site of the emergency, well before arrival at the hospital. In a departure from current practice, stroke patients could be differentially treated according to their ischemic or hemorrhagic etiology even in the prehospital phase of stroke management. Immediate diagnosis of cerebral ischemia and exclusion of thrombolysis contraindications enabled us to perform prehospital rt-PA thrombolysis as bridging to later intra-arterial recanalization in one patient. In a complementary patient with cerebral hemorrhage, prehospital diagnosis allowed immediate initiation of hemorrhage-specific blood pressure management and telemedicine consultation regarding surgery. Call-to-therapy-decision times were 35 minutes.

Conclusion

This preliminary study proves the feasibility of guideline-adherent, etiology-specific and causal treatment of acute stroke directly at the emergency site.     

Recanalization of Extracranial Internal Carotid Artery Occlusion after i.v. Thrombolysis for Acute Ischemic Stroke

Background

Although extracranial internal carotid artery (e-ICA) occlusion is a common pathology in patients undergoing intravenous thrombolysis for treatment of acute ischemic stroke, no data on e-ICA recanalization rate or potential effects on outcome are yet available.

Methods and Results

This study included 52 consecutive patients with e-ICA occlusion and ischemic stroke undergoing standard intravenous thrombolysis. The rate of e-ICA recanalization was 30.8% [95%CI, 18.2–43.3], documented at 3.5 [2.0–11.8] (median [IQR]) days after stroke, as compared to 8.6% [95%CI, 3.5–13.7] in a series of 116 consecutive patients with symptomatic e-ICA occlusion not undergoing thrombolysis (P<0.001 for difference). Functional outcome three months after stroke did not significantly differ for those with or without e-ICA recanalization following intravenous thrombolysis (modified Rankin scale ≤2: 31.3% vs. 22.2%, odds ratio 1.6 [95%CI, 0.4–5.9], P = 0.506). In patients with e-ICA occlusion of atherothrombotic origin, recanalization resulted in most instances in residual high-grade stenosis (13 of 14).

Conclusions

Recanalization of e-ICA occlusion after stroke thrombolysis occurred in about one third of patients. Although e-ICA recanalization had no significant effect on patient outcome, control sonography in the early days after thrombolysis is recommended for the detection of potential residual e-ICA stenosis.     

Warfarin anticoagulation exacerbates the risk of hemorrhagic transformation after rt-PA treatment in experimental stroke: therapeutic potential of PCC

Background

Oral anticoagulant therapy (OAT) with warfarin is the standard of stroke prevention in patients with atrial fibrillation. Approximately 30% of patients with cardioembolic strokes are on OAT at the time of symptom onset. We investigated whether warfarin exacerbates the risk of thrombolysis-associated hemorrhagic transformation (HT) in a mouse model of ischemic stroke.

Methods

62 C57BL/6 mice were used for this study. To achieve effective anticoagulation, warfarin was administered orally. We performed right middle cerebral artery occlusion (MCAO) for 3 h and assessed functional deficit and HT blood volume after 24 h.

Results

In non-anticoagulated mice, treatment with rt-PA (10 mg/kg i.v.) after 3 h MCAO led to a 5-fold higher degree of HT compared to vehicle-treated controls (4.0±0.5 µl vs. 0.8±0.1, p<0.001). Mice on warfarin revealed larger amounts of HT after rt-PA treatment in comparison to non-anticoagulated mice (9.2±3.2 µl vs. 2.8±1.0, p<0.05). The rapid reversal of anticoagulation by means of prothrombin complex concentrates (PCC, 100 IU/kg) at the end of the 3 h MCAO period, but prior to rt-PA administration, neutralized the exacerbated risk of HT as compared to sham-treated controls (3.8±0.7 µl vs. 15.0±3.8, p<0.001).

Conclusion

In view of the vastly increased risk of HT, it seems to be justified to withhold tPA therapy in effectively anticoagulated patients with acute ischemic stroke. The rapid reversal of anticoagulation with PCC prior to tPA application reduces the risk attributed to warfarin pretreatment and may constitute an interesting therapeutic option.     

Elevated Intracranial Pressure and Cerebral Edema following Permanent MCA Occlusion in an Ovine Model

Introduction

Malignant middle cerebral artery (MCA) stroke has a disproportionately high mortality due to the rapid development of refractory space-occupying cerebral edema. Animal models are essential in developing successful anti-edema therapies; however to date poor clinical translation has been associated with the predominately used rodent models. As such, large animal gyrencephalic models of stroke are urgently needed. The aim of the study was to characterize the intracranial pressure (ICP) response to MCA occlusion in our recently developed ovine stroke model.

Materials and Methods

30 adult female Merino sheep (n = 8–12/gp) were randomized to sham surgery, temporary or permanent proximal MCA occlusion. ICP and brain tissue oxygen were monitored for 24 hours under general anesthesia. MRI, infarct volume with triphenyltetrazolium chloride (TTC) staining and histology were performed.

Results

No increase in ICP, radiological evidence of ischemia within the MCA territory but without space-occupying edema, and TTC infarct volumes of 7.9+/-5.1% were seen with temporary MCAO. Permanent MCAO resulted in significantly elevated ICP, accompanied by 30% mortality, radiological evidence of space-occupying cerebral edema and TTC infarct volumes of 27.4+/-6.4%.

Conclusions

Permanent proximal MCAO in the sheep results in space-occupying cerebral edema, raised ICP and mortality similar to human malignant MCA stroke. This animal model may prove useful for pre-clinical testing of anti-edema therapies that have shown promise in rodent studies.     

In Vivo MR Imaging of Intraarterially Delivered Magnetically Labeled Mesenchymal Stem Cells in a Canine Stroke Model

Background

This study aimed to evaluate the feasibility of intraarterial (IA) delivery and in vivo MR imaging of superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs) in a canine stroke model.

Methodology

MSCs harvested from beagles’ bone marrow were labeled with home-synthesized SPIO. Adult beagle dogs (n = 12) were subjected to left proximal middle cerebral artery (MCA) occlusion by autologous thrombus, followed by two-hour left internal carotid artery (ICA) occlusion with 5 French vertebral catheter. One week later, dogs were classified as three groups before transplantation: group A: complete MCA recanalization, group B: incomplete MCA recanalization, group C: no MCA recanalization. 3×10⁶ labeled-MSCs were delivered through left ICA. Series in vivo MRI images were obtained before cell grafting, one and 24 hours after transplantation and weekly thereafter until four weeks. MRI findings were compared with histological studies at the time point of 24 hours and four weeks.

Principal Findings

Home-synthesized SPIO was useful to label MSCs without cell viability compromise. MSCs scattered widely in the left cerebral hemisphere in group A, while fewer grafted cells were observed in group B and no cell was detected in group C at one hour after transplantation. A larger infarction on the day of cell transplantation was associated with more grafted cells in the brain. Grafted MSCs could be tracked effectively by MRI within four weeks and were found in peri-infarction area by Prussian blue staining.

Conclusion

It is feasible of IA MSCs transplantation in a canine stroke model. Both the ipsilateral MCA condition and infarction volume before transplantation may affect the amount of grafted cells in target brain. In vivo MR imaging is useful for tracking IA delivered MSCs after SPIO labeling.     

Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

Background and Purpose

Microglia are resident immunocompenent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat.

Methods

Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR.

Results

HMO6 human microglial cells transplantion group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group.

Conclusions

Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin.     

FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia

Background

The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/Principal Findings

FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/Significance

In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.     

The GPVI-Fc Fusion Protein Revacept Improves Cerebral Infarct Volume and Functional Outcome in Stroke

Objectives

We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice.

Background

Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding.

Methods

Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery.

Results

Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept IV, compared to Fc only. 1 mg/kg Revacept IV applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen.

Conclusions

Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.     

Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms

Objective

Intra-arterial stem cell transplantation exerts neuroprotective effects for ischemic stroke. However, the optimal therapeutic time window and mechanisms have not been completely understood. In this study, we investigated the relationship between the timing of intra-arterial transplantation of allogeneic mesenchymal stem cells (MSCs) in ischemic stroke model in rats and its efficacy in acute phase.

Methods

Adult male Wistar rats weighing 200 to 250g received right middle cerebral artery occlusion (MCAO) for 90 minutes. MSCs (1×10⁶cells/ 1ml PBS) were intra-arterially injected at either 1, 6, 24, or 48 hours (1, 6, 24, 48h group) after MCAO. PBS (1ml) was intra-arterially injected to control rats at 1 hour after MCAO. Behavioral test was performed immediately after reperfusion, and at 3, 7 days after MCAO using the Modified Neurological Severity Score (mNSS). Rats were euthanized at 7 days after MCAO for evaluation of infarct volumes and the migration of MSCs. In order to explore potential mechanisms of action, the upregulation of neurotrophic factor and chemotactic cytokine (bFGF, SDF-1α) induced by cell transplantation was examined in another cohort of rats that received intra-arterial transplantation at 24 hours after recanalization then euthanized at 7 days after MCAO for protein assays.

Results

Behavioral test at 3 and 7 days after transplantation revealed that stroke rats in 24h group displayed the most robust significant improvements in mNSS compared to stroke rats in all other groups (p’s<0.05). Similarly, the infarct volumes of stroke rats in 24h group were much significantly decreased compared to those in all other groups (p’s<0.05). These observed behavioral and histological effects were accompanied by MSC survival and migration, with the highest number of integrated MSCs detected in the 24h group. Moreover, bFGF and SDF-1α levels of the infarcted cortex were highly elevated in the 24h group compared to control group (p’s<0.05).

Conclusions

These results suggest that intra-arterial allogeneic transplantation of MSCs provides post-stroke functional recovery and reduction of infarct volumes in ischemic stroke model of rats. The upregulation of bFGF and SDF-1α likely played a key mechanistic role in enabling MSC to afford functional effects in stroke. MSC transplantation at 24 hours after recanalization appears to be the optimal timing for ischemic stroke model, which should guide the design of clinical trials of cell transplantation for stroke patients.     

α-Lipoic Acid Reduces Infarct Size and Preserves Cardiac Function in Rat Myocardial Ischemia/Reperfusion Injury through Activation of PI3K/Akt/Nrf2 Pathway

Background

The present study investigates the effects and mechanisms of α-Lipoic acid (LA) on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in rat hearts subjected to in vivo myocardial ischemia/reperfusion (MI/R) injury.

Methodology/Principal Findings

Male adult rats underwent 30 minutes of ischemia followed by 3, 24, or 72 h of reperfusion. Animals were pretreated with LA or vehicle before coronary artery ligation. The level of MI/R- induced LDH and CK release, infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined and compared. Western blot analysis was performed to elucidate the mechanism of LA pretreatment. The level of inflammatory cytokine TNF-α released to serum and accumulated in injured myocardium as well as neutrophil accumulation in injured myocardium were also examined after MI/R injury. Our results reveal that LA administration significantly reduced LDH and CK release, attenuated myocardial infarct size, decreased cardiomyocytes apoptosis, and partially preserved heart function. Western blot analysis showed that LA pretreatment up-regulated Akt phosphorylation and Nrf2 nuclear translocation while producing no impact on p38MAPK activation or nitric oxide (NO) production. LA pretreatment also increased expression of HO-1, a major target of Nrf2. LA treatment inhibited neutrophil accumulation and release of TNF-α. Moreover, PI3K inhibition abolished the beneficial effects of LA.

Conclusions/Significance

This study indicates that LA attenuates cardiac dysfunction by reducing cardiomyoctyes necrosis, apoptosis and inflammation after MI/R. LA exerts its action by activating the PI3K/Akt pathway as well as subsequent Nrf2 nuclear translocation and induction of cytoprotective genes such as HO-1.     

Relationship between Blood Pressure and Outcomes in Acute Ischemic Stroke Patients Administered Lytic Medication in the TIMS-China Study

Objective

Increased blood pressure (BP) management following acute ischemic stroke (AIS) remains controversial. This study aimed to identify the association between BP and clinical outcomes in AIS patients administered lytic medication in the TIMS-China (thrombolysis implementation and monitor of acute ischemic stroke in China) database.

Methods

The sample comprised 1128 patients hospitalized within 4.5 hours (h) of AIS for intravenous recombinant tissue plasminogen activator (i.v. rt-PA) thrombolysis. Systolic BP (SBP) and diastolic BP (DBP) at baseline, 2 h and 24 h after treatment, and changes from baseline were analyzed. The study outcomes comprised a favorable outcome (modified Rankin Scale 0–1 at 90 days) and symptomatic intracerebral hemorrhage (SICH), analyzed using logistic regression, with low BP as the reference group.

Results

Lower BP (baseline, 2 h, and 24 h) was beneficial in AIS patients and significantly related to a favorable outcome (P<0.05). A substantial BP decrease at 24 h after rt-PA thrombolysis was significantly associated with a favorable outcome compared with a moderate BP decrease (P = 0.0298). A SBP >160 mmHg 2 h after rt-PA thrombolysis was significantly associated with SICH compared with a SBP <140 mmHg (P = 0.0238). An increase or no change (>25 mmHg) in SBP was significantly associated with SICH (P = 0.002) compared with a small SBP decrease (1–9 mmHg).

Conclusions

This study provides novel evidence that lower BP within the first 24 h is associated with a more favorable outcome and less frequent SICH in AIS patients administered lytic medication. Routine BP-lowering treatment should be considered in AIS patients following lytic medication.     

Reliability of Visual Assessment of Non-Contrast CT,CT Angiography Source Images and CT Perfusion in Patients with Suspected Ischemic Stroke

Background and Purpose

Good reliability of methods to assess the extent of ischemia in acute stroke is important for implementation in clinical practice, especially between observers with varying experience. Our aim was to determine inter- and intra-observer reliability of the 1/3 middle cerebral artery (MCA) rule and the Alberta Stroke Program Early CT Score (ASPECTS) for different CT modalities in patients suspected of acute ischemic stroke.

Methods

We prospectively included 105 patients with acute neurological deficit due to suspected acute ischemic stroke within 9 hours after symptom onset. All patients underwent non-contrast CT, CT perfusion and CT angiography on admission. All images were evaluated twice for presence of ischemia, ischemia with >1/3 MCA involvement, and ASPECTS. Four observers evaluated twenty scans twice for intra-observer agreement. We used kappa statistics and intraclass correlation coefficient to calculate agreement.

Results

Inter-observer agreement for the 1/3 MCA rule and ASPECTS was fair to good for non-contrast CT, poor to good for CT angiography source images, but excellent for all CT perfusion maps (cerebral blood volume, mean transit time, and predicted penumbra and infarct maps). Intra-observer agreement for the 1/3 MCA rule and ASPECTS was poor to good for non-contrast CT, fair to moderate for CT angiography source images, and good to excellent for all CT perfusion maps.

Conclusion

Between observers with a different level of experience, agreement on the radiological diagnosis of cerebral ischemia is much better for CT perfusion than for non-contrast CT and CT angiography source images, and therefore CT perfusion is a very reliable addition to standard stroke imaging.     

Effects of Acupuncture at GV20 and ST36 on the Expression of Matrix Metalloproteinase 2, Aquaporin 4, and Aquaporin 9 in Rats Subjected to Cerebral Ischemia/Reperfusion Injury

Background/Purpose

Ischemic stroke is characterized by high morbidity and mortality worldwide. Matrix metalloproteinase 2 (MMP2), aquaporin (AQP) 4, and AQP9 are linked to permeabilization of the blood-brain barrier (BBB) in cerebral ischemia/reperfusion injury (CIRI). BBB disruption, tissue inflammation, and MMP/AQP upregulation jointly provoke brain edema/swelling after CIRI, while acupuncture and electroacupuncture can alleviate CIRI symptoms. This study evaluated the hypothesis that acupuncture and electroacupuncture can similarly exert neuroprotective actions in a rat model of middle cerebral artery occlusion (MCAO) by modulating MMP2/AQP4/APQ9 expression and inflammatory cell infiltration.

Methods

Eighty 8-week-old Sprague-Dawley rats were randomly divided into sham group S, MCAO model group M, acupuncture group A, electroacupuncture group EA, and edaravone group ED. The MCAO model was established by placement of a suture to block the middle carotid artery, and reperfusion was triggered by suture removal in all groups except group S. Acupuncture and electroacupuncture were administered at acupoints GV20 (governing vessel-20) and ST36 (stomach-36). Rats in groups A, EA, and ED received acupuncture, electroacupuncture, or edaravone, respectively, immediately after MCAO. Neurological function (assessed using the Modified Neurological Severity Score), infarct volume, MMP2/AQP4/AQP9 mRNA and protein expression, and inflammatory cell infiltration were all evaluated at 24 h post-reperfusion.

Results

Acupuncture and electroacupuncture significantly decreased infarct size and improved neurological function. Furthermore, target mRNA and protein levels and inflammatory cell infiltration were significantly reduced in groups A, EA, and ED vs. group M. However, MMP2/AQP levels and inflammatory cell infiltration were generally higher in groups A and EA than in group ED except MMP2 mRNA levels.

Conclusions

Acupuncture and electroacupuncture at GV20 and ST36 both exercised neuroprotective actions in a rat model of MCAO, with no clear differences between groups A and EA. Therefore, acupuncture and electroacupuncture might find utility as adjunctive and complementary treatments to supplement conventional therapy for ischemic stroke.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

Lycium barbarum extracts protect the brain from blood-brain barrier disruption and cerebral edema in experimental stroke

Background and Purpose

Ischemic stroke is a destructive cerebrovascular disease and a leading cause of death. Yet, no ideal neuroprotective agents are available, leaving prevention an attractive alternative. The extracts from the fruits of Lycium barbarum (LBP), a Chinese anti-aging medicine and food supplement, showed neuroprotective function in the retina when given prophylactically. We aim to evaluate the protective effects of LBP pre-treatment in an experimental stroke model.

Methods

C57BL/6N male mice were first fed with either vehicle (PBS) or LBP (1 or 10 mg/kg) daily for 7 days. Mice were then subjected to 2-hour transient middle cerebral artery occlusion (MCAO) by the intraluminal method followed by 22-hour reperfusion upon filament removal. Mice were evaluated for neurological deficits just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, immunohistochemical analysis, and Western blot experiments. Evans blue (EB) extravasation was determined to assess blood-brain barrier (BBB) disruption after MCAO.

Results

LBP pre-treatment significantly improved neurological deficits as well as decreased infarct size, hemispheric swelling, and water content. Fewer apoptotic cells were identified in LBP-treated brains by TUNEL assay. Reduced EB extravasation, fewer IgG-leaky vessels, and up-regulation of occludin expression were also observed in LBP-treated brains. Moreover, immunoreactivity for aquaporin-4 and glial fibrillary acidic protein were significantly decreased in LBP-treated brains.

Conclusions

Seven-day oral LBP pre-treatment effectively improved neurological deficits, decreased infarct size and cerebral edema as well as protected the brain from BBB disruption, aquaporin-4 up-regulation, and glial activation. The present study suggests that LBP may be used as a prophylactic neuroprotectant in patients at high risk for ischemic stroke.     

K-134, a Phosphodiesterase 3 Inhibitor,Prevents Brain Damage by Inhibiting Thrombus Formation in a Rat Cerebral Infarction Model

Background

K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol.

Objectives

This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model.

Methods and Results

We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (n = 12, 87.5±5.6 vs. 126.8±7.5 mm³, P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol.

Conclusions

These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability.     

Nox2 oxidase activity accounts for the oxidative stress and vasomotor dysfunction in mouse cerebral arteries following ischemic stroke

Background and Purpose

Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase.

Methods

Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2^-/-) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N ^ω-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting.

Results

Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2^-/- mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2^-/- mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2^-/- mice. Vascular CD45 levels were unchanged by ischemia-reperfusion.

Conclusions

Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.     

Functional Real-Time Optoacoustic Imaging of Middle Cerebral Artery Occlusion in Mice

Background and Purpose

Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.

Materials and Methods

The middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points.

Results

Post ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit.

Conclusion

The intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area.     

Intravenous Thrombolysis with Recombinant Tissue Plasminogen Activator for Ischemic Stroke Patients over 80 Years Old: The Fukuoka Stroke Registry

Objectives

The benefit of intravenous recombinant tissue plasminogen activator (rt-PA) therapy for very old patients with acute ischemic stroke remains unclear. The aim of this study was to elucidate the efficacy and safety of intravenous rt-PA therapy for patients over 80 years old.

Methods

Of 13,521 stroke patients registered in the Fukuoka Stroke Registry in Japan from June 1999 to February 2013, 953 ischemic stroke patients who were over 80 years old, hospitalized within 3 h of onset, and not treated with endovascular therapy were included in this study. Among them, 153 patients were treated with intravenous rt-PA (0.6 mg/kg). For propensity score (PS)-matched case-control analysis, 148 patients treated with rt-PA and 148 PS-matched patients without rt-PA therapy were selected by 1∶1 matching with propensity for using rt-PA. Clinical outcomes were neurological improvement, good functional outcome at discharge, in-hospital mortality, and hemorrhagic complications (any intracranial hemorrhage [ICH], symptomatic ICH, and gastrointestinal bleeding).

Results

In the full cohort of 953 patients, rt-PA use was associated positively with neurological improvement and good functional outcome, and negatively with in-hospital mortality after adjustment for multiple confounding factors. In PS-matched case-control analysis, patients treated with rt-PA were still at lower risk for unfavorable clinical outcomes than non-treated patients (neurological improvement, odds ratio 2.67, 95% confidence interval 1.61–4.40; good functional outcome, odds ratio 2.23, 95% confidence interval 1.16–4.29; in-hospital mortality, odds ratio 0.30, 95% confidence interval 0.13–0.65). There was no significant association between rt-PA use and risk of hemorrhagic complications in the full and PS-matched cohorts.

Conclusions

Intravenous rt-PA therapy was associated with improved clinical outcomes without significant increase in risk of hemorrhagic complications in very old patients (aged>80 years) with acute ischemic stroke.