Embolic Middle Cerebral Artery Research Articles

Abstract WP113: Dose-response and Therapeutic Time-window of Compound 21: a Randomized Preclinical Trial in Rat Model of Thromboembolic Stroke

Background: We and others have previously shown promising results with the use of the angiotensin type 2 receptor agonist, compound 21 (C21), in experimental stroke. Here we aimed to determine the best dose and time window for C21 in a clinically relevant embolic stroke model. Methods: This study was conducted as a translational, randomized, controlled, blinded preclinical trial with the guidance of our biostatistician. Male Wistar rats (8-9 weeks old) were subjected to embolic middle cerebral artery occlusion (eMCAO). For the dose-response study, animals received C21 (0.01, 0.03 and 0.06 mg/kg/d) or saline (orally) for 5 days, with the first dose given IV at 3 h post-eMCAO. For the time-window study, the optimal dose of C21 was initiated at 3, 6 or 24 h post-eMCAO and daily thereafter, for 5 days. Behavioral outcomes (Bederson, paw grasp and grip strength) were blindly assessed at days, 1, 3, 5 and 7. Rats were then sacrificed and their brains collected for infarct size and vascular density analyses. Results (Table): In the dose-response study, repeated-measures ANOVA showed significant (p < 0.05) behavioral improvement with the low dose (0.01 mg/kg). This dose was associated with higher vascularity in the ischemic penumbra, detected by laminin staining, compared to saline treated controls (65±6 vs 46±7 p < 0.05). In the time-window study, the 0.01 mg/kg dose displayed non-statistically significant improvements when given 3 h and 6 h after eMCAO. There were no differences between doses or time-windows for either % infarct size or tissue loss. Conclusions: C21 given at 0.01 mg/kg/d was effective in improving behavioral dysfunction after embolic stroke when administered within 6 h. Compound C21 shows promise as a potential therapeutic agent and should be examined for safety and efficacy in clinical trials for ischemic stroke.

Abstract WP100: Thymosin β4 for the Treatment of Sub-Acute Stroke in Aged Rats

Introduction: Thymosin β4 (Tβ4) is a 5K peptide which influences cellular migration by inhibiting organization of the actin-cytoskeleton. Tβ4 has neurorestorative properties and is a potential candidate for the treatment of sub-acute stroke. Previous research demonstrated that Tβ4 improved neurological outcome in a young (3 months) rat model of embolic stroke. Hypothesis: We hypothesized that Tβ4 would improve neurological outcome in an aged rat model of embolic stroke when administered 24 hours after embolic stroke. Methods: Aged Male Wistar rats (Charles River, France 18-21 months) were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were randomized to receive Tβ4 (12 mg/kg, Regenerx Biopharmaceuticals, Inc.) or control 24 hrs after MCAo and then every 3 days for 4 additional doses. The dose of 12 mg/kg was the maximal dose of Tβ4 that showed functional improvement in a young rat model of embolic stroke. Functional tests were performed weekly. The rats were sacrificed 56 days after MCAo and lesion volumes measured. Generalized Estimating Equations were used to compare the treatment effect on functional recovery and t-test for lesion volumes. Results and Conclusions: Twenty-three rats were included in the study: control group (n=12) and Tβ4 group (n=11). After randomization, there were three deaths in both the control and Tβ4 groups. The Tβ4 treatment reduced infarct volume by more than 50% (12.8% ± 9.3%, mean ± SE, p<0.05) compared to the control group (26.0% ± 4.3%). However, Tβ4 did not show improvement in functional outcome compared to control. Lesion volumes in the treated group showed a correlation to functional testing of (R=0.85, p<0.05), whereas no correlation was observed in the control group (R=0.19). Conclusions: The Tβ4 treatment of stroke aged animals significantly reduces infarct volume compared to vehicle treated stroke. Dissociation between infarct volume and functional outcome in the control group suggests, a ceiling effect of these functional tests in aged animals may obscure proper evaluation of functional outcome between the Tβ4 and control groups. Additional studies and more sensitive behavioral tests are needed to examine the dissociation between infarct volume and functional outcomes in aged ischemic rats.

Abstract 169: Human Dose tPA Improves Functional Outcomes in Both Sexes in Embolic Model of Stroke: Differential Effect on Hemorrhagic Transformation

Tissue plasminogen activator (tPA) is the only FDA-approved therapeutic agent for the treatment of acute ischemic stroke. The widespread use of tPA is still limited by the fear of hemorrhagic transformation (HT) and underlying mechanisms are actively being pursued in preclinical studies. However, experimental models use a 10 times higher dose of tPA than the clinical dose (10 mg/kg) and mostly employ only male animals. In this translational study, we hypothesized that low dose tPA would achieve clot lysis, decrease neurovascular injury and improve functional outcomes in both sexes. Aged-matched male and female Wistar rats (n=5-7) have been treated with or without tPA (1 mg/kg, i.v.) at 90 min after embolic middle cerebral artery occlusion with a fibrin-rich humanized clot. The neurological deficiency (Bederson score and adhesive removal test -ART), infarct size, HT index, and edema ratio were assessed 3 days after surgery (Table). Compared to male rats, female rats had smaller infarct size and better functional outcomes as previously reported in the literature. tPA decreased infarct size in both sexes. tPA reduced edema in males with no effect in females. While there was no difference in HT between males and females without tPA, HT was less in the female + tPA group. Functional outcomes, especially ART, were significantly improved with tPA in both sexes. These data suggest that 1) thrombolysis with a low dose tPA is effective in improving short term outcomes in both sexes, and 2) better functional outcomes in females are further enhanced with tPA. Additional studies are in progress to explore long term effects and the impact of therapeutic window, age and sex steroids on outcomes as well as the underlying mechanisms contributing to less HT in females.

Abstract WP102: Cerebrolysin Improves Neurological Outcome in Rats After Acute Stroke: a Prospective, Randomized, Blinded, Placebo-controlled Study

Background and Purpose: Cerebrolysin is a mixture of neuropeptides and free amino acids that exert potent neuroprotective and neurorestorative properties in experimental models of stroke. We conducted a prospective, randomized, double-blind and placebo controlled dose-response study of Cerebrolysin in both male and female rats after embolic stroke. Methods: Randomization schemas were generated using nQuery3.0. Male and female Wistar rats (3-4 months) were subjected to embolic middle cerebral artery occlusion (MCAO). After confirming successful stroke with a 5 point neurological scale (Longa scale) at 3h after MCAO, ischemic rats were treated with Cerebrolysin at doses of (0.8, 2.5, 5.0, 7.5ml/kg) and saline based on the randomization schema 4h after MCAO and continued daily for a total of 10 consecutive days. The primary outcome was neurologic outcome measured by adhesive removal test, foot-fault test, and modified neurological severity score at day 28, lesion volume and mortality were secondary and safety outcomes, respectively. Results: There was a significant dose effect of Cerebrolysin on neurological functional improvement 28 day after MCAO. Cerebrolysin at a dose of ≥ 2.5ml/kg significantly (P<0.001) improved neurological outcome (Mean Estimate with 95%CL): 0.8ml/kg: 6.2 (-6.0/18.4), 2.5ml/kg: -28.9 (-41.6/-16.2), 5.0ml/kg: -33.4 (-45.0/-21.7), 7.5ml/kg: -36.3 (-48.2/-24.4). Higher doses (≥2.5ml/kg) resulted in better recovery; however, differences between effective doses were not significant. Treatment with 5ml/kg reduced lesion volume (19.5±8.9%) compared with saline treated rats (27.7±11.9%, P=0.016). No treatment gender interactions were found and there were no differences on mortality rate. Conclusion: Cerebrolysin in a dose-dependent manner significantly improved neurological outcomes in ischemic rats. Cerebrolysin at a dose of 5ml/kg reduced infarct volume. Our data on Cerebrolysin efficacy demonstrate the feasibility of a preclinical study setup following a randomized, placebo controlled, and blinded design with a clinical relevant treatment scheme.

Cerebrolysin dose-dependently improves neurological outcome in rats after acute stroke: A prospective, randomized, blinded, and placebo-controlled study.

Cerebrolysin is a mixture of neuropeptides and free amino acids that is clinically used for the treatment of stroke. To further standardize treatment schemes, we assessed the dose response of Cerebrolysin on sensorimotor outcome in a rat model of ischemic stroke. This study was a prospective, blinded, placebo-controlled, preclinical experiment. Male and female Wistar rats, subjected to embolic middle cerebral artery occlusion, were randomly treated with Cerebrolysin doses of 0.8, 2.5, 5.0, 7.5 ml/kg or placebo, 4 h after middle cerebral artery occlusion for a total of 10 consecutive days. The primary outcome was neurologic improvement at day 28, lesion volume, mortality, and animal weight were secondary and safety outcomes, respectively. There was a significant (p < 0.001) dose effect of Cerebrolysin on neurological outcome. Cerebrolysin at a dose of ≥ 2.5 ml/kg significantly (p < 0.001) improved neurological outcome (Mean Estimate (95% CL): 0.8 ml/kg: 6.2 (-6.0/18.4), 2.5 ml/kg: -28.9 (-41.6/-16.2), 5.0 ml/kg: -33.4 (-45.0/-21.7), 7.5 ml/kg: -36.3 (-48.2/-24.4). Higher doses (≥ 2.5 ml/kg) resulted in better recovery; however, differences between effective doses were not significant. Treatment with 5 ml/kg reduced lesion volume (p = 0.016). No treatment gender interactions were found and there were no differences in death or weight loss. Collectively, these data on Cerebrolysin efficacy demonstrate the feasibility of a preclinical study setup following a randomized, placebo-controlled, and blinded design with a clinical relevant treatment scheme. Cerebrolysin at doses of ≥ 2.5 ml/kg improved functional outcome and at a dose of 5 ml/kg reduced infarct volume.

Embolic Middle Cerebral Artery Occlusion from Innominate Artery Stenosis: Successful Mechanical Recanalisation

Embolic complications resulting from significant innominate artery stenosis (IAS) is a rare pathology. We present a clinical case of IAS with subsequent middle cerebral artery (MCA) occlusion detected with cerebral computed tomography and neurovascular ultrasound and successfully treated using endovascular embolectomy within 4 hours after symptom onset. The patient recovered well. Additional IAS endarterectomy days later showed fresh thrombi on the vessel wall, and resulted in complete normalisation of cerebral blood flow. This is to the best of our knowledge the first case of MCA embolectomy from an emboligenic IAS. Although not used in the successful embolectomy trials in the last years, neurosonography was crucial to guide the revascularisation therapy and the hemodynamic compromise in IAS might have led to ischemic preconditioning and thus better outcome upon revascularisation. Endovascular embolectomy in patients with IAS and MCA occlusion must not necessarily need stent angioplasty of the IAS in the acute stroke phase thus avoiding double platelet therapy upon cerebral reperfusion. Given the rareness of the disease, an international registry of similar cases may help to better understand the disease and guide acute interventions.

A novel embolic middle cerebral artery occlusion model induced by thrombus formed in common carotid artery in rat

Stroke is a major cause of death and disability worldwide. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental stroke models were critical for preclinical research. However, commonly used embolic stroke models are reluctant to mimic the clinical situation and not suitable for thrombolytic timing studies. In this paper, we established a standard method for producing a rat embolic stroke model with autologous thrombus formed within the common carotid artery (CCA) by constant galvanic stimulation. Then the thrombus was shattered and channeled into the origin of the MCA and small (lacunar) artery. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured at 2, 4 and 6h postischemia. This model developed a predictable infarct volume (38.37±2.88%) and gradually reduced blood flow (20% of preischemic baselines) within the middle cerebral artery (MCA) territory. The thrombus occluded in the MCA was able to be lysed by a tissue-type plasminogen activator (t-PA) within 4h postischemia. The techniques presented in this paper would help investigators to overcome technical problems for stroke research.

Effect of helicopter transport on neurological outcomes in a mouse model of embolic stroke with reperfusion: AIR-MICE pilot study.

Patients often suffer a stroke at a significant distance from a center capable of delivering endovascular therapy, thus requiring rapid transport by helicopter emergency medical services while receiving a recombinant tissue plasminogen activator infusion that was initiated locally. But little is known about how a helicopter flight may impact the safety and efficacy of recombinant tissue plasminogen activator-induced reperfusion and patient outcomes. To establish a new animal method to address with fidelity the safety and overall effect of helicopter emergency medical services during thrombolysis. Prospective randomized open blinded end-point study of an actual helicopter flight exposure. Adult C57BL/6 male mice were treated with a 10 mg/kg recombinant tissue plasminogen activator infusion two-hours after an embolic middle cerebral artery occlusion. Mice were randomized in pairs to simultaneously receive the infusion during a local helicopter flight or in a ground hangar. Eighteen mice (nine pairs) were analyzed. The paired t-test analysis showed nonsignificant smaller infarction volumes in the helicopter-assigned animals (mean pair difference 33 mm(3) , P = 0·33). The amount of hemorrhagic transformation between the helicopter and ground groups was 4·08 vs. 4·56 μl, respectively (paired t-test, P = 0·45). This study shows that helicopter emergency medical services do not have an inherent adverse effect on outcome in a mouse model of ischemic stroke with reperfusion. These results endorse the safety of the practice of using helicopter emergency medical services in stroke patients. The observed potential synergistic effect of helicopter-induced factors, such as vibration and changes in altitude, with reperfusion merits further exploration in animal experimental models and in stroke patients.

The impact of electroacupuncture on the expression of AQP4 and CLN5 in the blood-brain barrier after cerebral ischemia and reperfusion

Objective To observe the effect of electroacupuncture (EA) pretreatment of different durations on the expressions of AQP4 and CLN5 in the blood-brain barrier after cerebral ischemia and reperfusion Methods Sixty-four male Sprague-Dawley (SD) rats were randomly divided into a model group, a sham operation group, a 7-day EA-pretreatment group (EAP-7 group) and a 15-day EA-pretreatment group (EAP-15 group), each of 16. After EA-pretreatment on the baihui and shuigou acupoints for 7 days or 15 days, a model of unilateral middle cerebral artery embolism (MCAO) model was induced in the rats of the model, EAP-7 and EAP-15 group using a modified Longa method. In the sham operation group the carotid artery was separated without middle cerebral artery embolism. The reperfusion was begun 90 min after the MCAO modeling. Immunohistochemical methods and fluorescence quantitative PCR were applied to detect the expression of AQP4, CLN5 and their mRNAs expression in the blood brain barrier (BBB) 24h after the operation. Results Compared with the sham operation group, the expression of AQP4 positive cells and AQP4 mRNA in the BBB in the other three groups had increased significantly, while the expression of CLN5 positive cells and CLN5 mRNA was significantly less. Compared with the model group, the expression of AQP4 positive cells and AQP4 mRNA in the EAP-7 and EAP-15 groups was significantly reduced, while the expression of CLN5 positive cells and CLN5 mRNA was significantly increased. Moreover, the expression of AQP4 positive cells and AQP4 mRNA in the EAP-15 group were significantly higher than in the EAP-7 group, while the expression of CLN5 positive cells and CLN5 mRNA were significantly lower. Conclusions EA-pretreatment on the baihui and shuigou acupoints can restrain the expression of AQP4-positive cells and AQP4 mRNA and promote that of CLN5-positive cells and CLN5 mRNA in the BBB after cerebral ischemia and reperfusion, at least in rats. The BBB protection effect is better when the EA-pretreatment lasts longer. The mechanisms of cerebral ischemia tolerance may be related to the regulation of AQP4, CLN5 and their mRNAs in the blood-brain barrier after cerebral injury. Key words: Acupuncture; Cerebral ischemia; Reperfusion; Blood-brain barrier; AQP4; CLN5

Abstract W P101: Combination of MST-188 and Tissue Plasminogen Activator Extends the Therapeutic Window of Stroke

Background and Purpose: The utilization of tissue plasminogen activator (tPA) for stroke treatment is hampered by its narrow therapeutic window and hemorrhagic complication. MST-188 (Vepoloxamer) is a proprietary tri-block copolymer with rheological and membrane-protective properties. The current study investigated whether MST-188 enhances the therapeutic effect of tPA on ischemic damage in a rat model of embolic focal cerebral ischemia. Methods: Male Wistar rats (n=10) subjected to embolic middle cerebral artery occlusion (MCAO) were treated post stroke with the combination of MST-188 (300 mg/kg at 3.5h, IV; followed by a second dose of 2,000 mg/kg at 9.5h, IP) and tPA (10 mg/kg at 4h, IV). Ischemic rats treated with tPA (10 mg/kg at 4h, IV) alone, or saline were used as control groups (n=10/group). An array of behavioral tests were performed at 1 and 7 days after MCAO. Ischemic lesion volume was measured 7 days after MCAO. Results: All rats exhibited neurological deficits measured by the Longa scale 30 min after MCAO. Treatment with tPA alone failed to reduce infarct volume and neurological functional deficits compared with saline treated rats. However, MST-188 in combination with tPA significantly (p&lt;0.05) reduced neurological functional deficits measured by the adhesive removal test (90±29 at 1d and 63±39 seconds at 7d) and modified neurological severity score (mNSS, 9±2 at 1d and 6±3at 7d) compared with rats treated with tPA alone (116±9 at 1d and 96±17 seconds at 7d by adhesive removal test, 11±1 at 1d and 9±1 at 7d by mNSS) and saline (113±12 at 1d and 95±16 seconds at 7d by adhesive removal test, 11±1 at 1d and 8±1 at 7d by mNSS). Histopathological analysis showed that the combination of MST-188 with tPA treatment significantly reduced lesion volume (21±10%, p&lt;0.05) compared with rats treated with tPA alone (35±12%) and saline (34±11%). No significant differences of the incidence of gross hemorrhage were detected among groups. Conclusions: MST-188 enhances the therapeutic effect of tPA without increasing the incidence of hemorrhagic transformation when tPA is administered 4h after embolic MCAO.

Abstract W MP52: Treatment With N-acetyl-seryl-aspartyl-lysyl-proline Improves Neurological Functional Recovery in Diabetic Middle Aged Rats After Embolic Stroke

Background and Purpose: Diabetes mellitus (DM) is a common metabolic disease among middle-aged and older populations, which leads to an increase of stroke incidence and poor stroke recovery. The present study investigated the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on stroke recovery in middle aged DM rats after embolic stroke. Methods: Male Wistar rats at age of 13 months were subjected to nicotinamide (NTM) and streptozotocin (STZ) injection . Rats with confirmed hyperglycemia status were subjected to embolic middle cerebral artery occlusion (MCAO) 30d after STZ-NTM injection. AcSDKP (2.4mg/kg) or saline were subcutaneously infused for 3d starting at 1d after stroke (n=12/group). Results: AcSDKP treatment significantly improved neurological functional recovery measured by adhesive removal test, foot-fault test, and modified neurological severity score starting at 21d after MCAO compared with saline treated rats. In addition, AcSDKP substantially reduced spatial learning deficits measured by the Morris water maze (41±4 vs 33±3% of time spent in the correct quadrant) 35d after MCAO. AcSDKP treatment significantly reduced vascular and parenchymal fibrin deposition (43±9 vs 92±11/mm 2 ) in the ipsilateral hippocampus and peri-infarct corpus callosum (CC), which was associated with reduction of IL-1 receptor associated kinase-1(IRAK1) immunoreactive vessels (95±13 vs 47±11/mm 2 ) 7d after MCAO (n=4/group). AcSDKP treatment did not reduce infarct volume, but robustly increased myelinated axonal density (14±4 vs 8±3% in hippocampus, 36±6 vs 27±7% in CC) and oligodendrocyte density (71±18 vs 46±16/mm 2 in hippocampus, 526±113 vs 394±107/mm 2 in CC) 35d after MCAO (n=8/group). In vitro, high glucose increased protein level of IRAK1 and NF-κB in primary cerebral endothelial cells, whereas AcSDKP suppressed high glucose-increased IRAK1 and NF-κB protein levels by 50%. Conclusions: AcSDKP promotes ischemic brain repair by reducing neurovascular damage in the hippocampus and white matter, which likely contributes improvement of neurological functional recovery in middle-aged DM rats. IRAK1/NF-κB signals suppressed by AcSDKP may be involved these processes.

Abstract T P235: Assessing the Impact of Rotorcrafts in a Model of Induced Cerebral Embolism (AIR-MICE)

Objective: Stroke is a time-dependent emergency that often requires emergent helicopter evacuation, a critical early setting for which little is known about the positive or deleterious effect of altitude change, noise, acceleration and vibration. While many of these factors could worsen ischemic injury, the low frequency vibrations might augment clot lysis and enhance rtPA treatment. The objective of this study is to measure the feasibility, safety and potential effect of helicopter transport during thrombolysis in an animal model. Methodology: Prospective, randomized, open intervention and blinded end-point pilot study that combines a controlled experimental model with a real life helicopter flight exposure. Subjects were adult C57BL/6 male mice that simultaneously underwent an embolic middle cerebral artery occlusion (eMCAO) in pairs. Each pair of mice were randomized to receive 10 mg/kg of rtPA at two hours after eMCAO either during a one hour local flight in a MI2 turbine helicopter, or to simultaneously receive the same rtPA infusion while waiting in an airport hangar. Mice were sacrificed at 24 h. Primary outcome measure was infarct volume. Secondary outcome was neurological scores. Results: Eighteen mice (9 pairs) were analyzed. Lower infarction volumes were seen in the helicopter assigned group in 6/9 pairs. The mean infarction volumes were 246.6 mm 3 (SD 154) for ground group and 213.6 mm 3 (SD 170) for the helicopter group. A paired t-test analysis showed lesser infarction volumes in the helicopter group (mean difference 33 mm 3 , p=0.33), a difference that was not influenced when adjusting for ambient temperature and atmospheric pressure. The neurological scores were similar in the ground and helicopter groups (1.28 vs. 1.22, p=0.88). Conclusion: Randomized trials of the effect of helicopter transport in a mouse model of eMCAO are feasible, and desirable to test the effect of neuroprotective therapies in this unique setting. These results endorse the safety of aerial transport, which might be associated with lower infarction volumes. The potential synergist effect of vibration in the rotorcraft with reperfusion needs to be explored further.

Abstract T P232: Combination Therapy With Taurine Plus Tissue Plasminogen Activator in a Rat Model of Embolic Stroke

Background: Tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke, but it has narrow therapeutic window (within 4.5h) and increased risks of intracerebral hemorrhage. Taurine, an endogenous 2-aminoethansulfolic amino acid, exhibits a plethora of physiological functions. It has been found to protect against ischemic brain injury in animal models of stroke by diverse mechanisms. Strikingly, published data show that the therapeutic window of taurine was at least 8h after the onset of ischemic stroke, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated whether combination therapy with taurine plus tPA can block hemorrhagic transformation and thus extend the time window of tPA after delayed tPA treatment for ischemic stroke. Methods and Results: Male adult rats (330-380g) were subjected to embolic middle cerebral artery occlusion and then randomized into different treatment groups: saline injected at 4h; taurine (50 mg/kg) at 4h, tPA (10 mg/kg) at 6h, and combination therapy with taurine at 4h plus tPA at 6h after the onset of ischemia. Delayed 6-hour tPA did not decrease infarction but instead worsened brain hemorrhage. Combining taurine with delayed 6-hour tPA reduced tissue infarction, attenuated blood-brain barrier disruption by reduced degradation of tight junction proteins (claudin-5 and collagen IV), and ameliorated brain hemorrhage. These protective effects are correlated with decreased brain matrix metallopeptidase-9 activity possibly through inactivation of NF-kB and CD147 (a potent inducer of MMPs) in the postischemic brain, particularly in the cerebral microvessels (using structurally intact microvessels isolated from the brain). Conclusion: The present study demonstrates for the first time that combination therapy with taurine may attenuate delayed tPA-associated hemorrhagic transformation and extend tPA treatment time windows in a clinically relevant thromboembolic stroke model. Inhibition of MMP-9 in brain microvessels via inactivation of NF-κB and CD147 signaling may underlie the protective mechanisms of taurine.

Combination of Ginsenoside Rg1 and Bone Marrow Mesenchymal Stem Cell Transplantation in the Treatment of Cerebral Ischemia Reperfusion Injury in Rats

Background/Aims: The present study aims to explore the protective role and mechanism of ginsenoside Rg1 combined with bone marrow mesenchymal stem cell (BMSC) transplantation for cerebral ischemia reperfusion injury (CIRI) in rat brain. Methods: One hundred twenty male SD rats were randomly divided into a sham group, an Ischemia Reperfusion (IR) group, an IR group treated with BMSC transplantation (IR+BMSCs), an IR group treated with Rg1 (IR+Rg1), and an IR group treated with BMSC transplantation and Rg1 (IR+Rg1+BMSCs). To establish a CIRI model, right middle cerebral artery embolization was used. The neurological score, 2,3,5-triphenyltet-razolium chloride monohydrate (TTC) staining and brain water content were detected to assess the treatment efficiency. HE staining and TUNEL were used to explore the pathologic changes and apoptosis. To explore the protein levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), immunofluoresence was utilized. Western blotting was used to explore apoptosis-related proteins such as Bcl-2 and Bax. Results: Compared with the sham group, the IR group demonstrated obvious ischemic changes, such as significant neurologic defects and enhanced brain water content. The Rg1 treatment resulted in an obvious decrease in cell apoptosis and improved ischemic conditions. By BMSC transplantation, the transplanted cells could be differentiated into neurons and glial cells, which also improved cerebral ischemia. More importantly, the IR+Rg1+BMSCs group showed the best treatment efficiency with reduced cell apoptosis and better cerebral recovery. Conclusions: The Rg1 treatment resulted in an obvious decrease in cell apoptosis, while the transplanted cells could be differentiated into neurons and glial cells, which also improved cerebral ischemia.

Effect and mechanism of Guhong injection against cerebral ischemia reperfusion injury

To explore the effect and mechanism of Guhong injection against cerebral ischemia reperfusion injury, SD rats were randomly divided into the sham-operated group, the model group, the nimodipine group, and high, medium and low-dose Guhong injection groups, with 10 rats in each group. The middle cerebral artery embolization (MCAO) model was established to observe neurological deficit symptoms, infarct volume, SOD activity, MDA content, GSH-Px and CAT activity in rats, as well as the contents of t-PA, PAI, TXB2 and 6-keto-PGF1α in serum. The results showed that Guhong injection could obviously promote the recovery of neurological deficit symptoms, narrow the brain infarct volume in rats after surgery, significantlyincrease the activities of SOD, GSH-Px and CAT and decrease the content of MDA. Meanwhile, it also could obviously increase the contents of t-PA and 6-keto-PGF1α and decrease the contents of PAI and TXB2 in serum, indicating that Guhong injection have better antioxidant and antithrombus effects, as well as a significant protective effect against cerebral ischemia reperfusion injury in rats.

P.1.g.001 Neuroprotective efficacy of coenzyme Q10 in rat model of transient focal ischemia

Thymosin β4 (Tβ4) is a developmentally expressed 43-amino acid peptide that inhibits organization of the actin-cytoskeleton by sequestration of G-actin monomers. Tβ4 improves cardiac function after myocardial infarction in adult mice and promotes healing properties in both dermal and corneal wounds. We tested the hypothesis that Tβ4 improves functional neurological outcome in a rat model of embolic stroke. Experimental Procedures: Male Wistar rats (n=18) were subjected to embolic middle cerebral artery occlusion (MCAo). Tβ4 (6 mg/kg, IP) was administered 24 h after MCAo and then every 3 days for four additional doses (n=9). Rats treated with saline were used as a control (n=9). The adhesive-removal test (ART) and modified Neurological Severity Score (mNSS) were performed to measure functional outcome. Rats were sacrificed 56 days after MCAo. Immunostaining was performed with antibodies against NG-2 (chondroitin sulfate proteoglycan), CNPase (2”, 3”-cyclic nucleotide 3'-phosphodiesterase) to detect immature and mature oligodendrocytes. Neurofilament-H (NF-H) antibodies were used to detect axons while myelinated axons were identified with Bielschowsky/Luxol (B/L) Blue staining. EBA (endothelial barrier antigen) was used for detection of mature vessels. Results: Ischemic rats treated with Tβ4 demonstrated a significant overall improvement (P<0.01) in the ART and the mNSS when compared to controls. Significant improvement was observed beginning at 14 and 35 days, respectively. Lesion volumes showed no significant differences between the two groups. Treatment with Tβ4 increased myelinated axons and increased vessel density in the ischemic boundary (P<0.05) and augmented remyelination which was associated with an increase of oligodendrocyte progenitor cells (OPCs) and myelinating oligodendrocytes (P<0.05). Conclusions: The present study suggests that Tβ4 improves neurological functional outcome after embolic stroke in rats. Axonal remodeling from mobilization of OPCs is proposed as contributing to Tβ4 induced functional improvement.

Potential advantages of Chinese medicine Taohong Siwu Decoction () combined with tissue-plasminogen activator for alleviating middle cerebral artery occlusion-induced embolic stroke in rats.

To investigate whether combination treatment with Taohong Siwu Decoction (, TSD) and recombinant tissue-type plasminogen activator (rt-PA) potentiate in reducing infarct volume and alleviate thromboembolic stroke in an in vivo rat model. Adult male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAO) and treated with rt-PA (4 and 8 mg/kg) alone (n=5), TSD [0.7 g/(kg·day)] alone (n=5), combination of rt-PA and TSD, 24 h after stroke. Rats were sacrificed at 14 days after treatment and lesion volumes were measured. To investigate the underlying mechanism of neuroprotective effect of the combination treatment, cleaved caspase-3, tumor necrosis factor alpha (TNF-α), hypoxia-inducible factor (HIF)-1α, and inducible nitric oxide synthase (iNOS) immunostaining were performed. Combination treatment significantly reduced infarct volume of cerebral ischemic regions compared with treatment of rt-PA and TSD alone and that of the saline control group (P<0.01). A combined treatment of rt-PA (4 mg/kg) with TSD [0.7 g/(kg·day)] significantly increased cerebral blood flow in a time (100 and 120 min) dependent manner (P<0.05). Interestingly, despite treatment of rt-PA (4 mg/kg) alone significantly reduced the expressions of HIF-1α, TNF-α, and iNOS in ischemic regions, reduction of these expressions were more potentiated when combined with TSD (P<0.05). Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared with the MCAO group (P<0.01). A combination of low-dose rt-PA and TSD after embolic stroke reduced infarct volume, improved cerebral blood flow and provided neuroprotection and these effects were associated with reduction of apoptosis and attenuation of HIF-1α, TNF-α and iNOS expression. These results provide a positive contribution to better understand the therapeutic value of the combination of TSD with rt-PA in ischemic stroke and may support further clinical evaluation.

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

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.

Larger A1/M1 diameter ratio predicts embolic anterior cerebral artery territorial stroke.

In contrast to middle cerebral artery territory strokes, anterior cerebral artery strokes (ACAS) occur rarely. The low frequency of ACAS, in relation to middle cerebral artery territory strokes, may be explained by differences in ACA and middle cerebral artery anatomy influencing their respective flow-directed embolism rates. We aimed to determine whether variability in ACA anatomy, and in particular A1 segment diameter, is associated with embolic ACAS. Consecutive patients admitted to Boston Medical Center with embolic ACAS were reviewed. Ipsilateral and contralateral A1 diameters, M1 diameters, and terminal internal carotid artery bifurcation angles were measured from computed tomographic angiography and MRI angiography images. We compared these measurements between cases of ACAS and consecutive cases of embolic middle cerebral artery territory strokes. The study comprised 55 individuals (27 ACAS, 28 middle cerebral artery territory strokes) with mean age of 69 years. In multivariate regression analysis, larger ipsilateral A1 diameters (odds ratio per 1 mm increment: 8.5; 95% confidence interval, 1.4-53.3) and ipsilateral A1/M1 diameter ratio (odds ratio per 10% increment: 1.8; 95% confidence interval, 1.2-2.9) were associated with ACAS, whereas larger ipsilateral M1 diameters was protective for ACAS (odds ratio per 1 mm increment: 0.8; 95% confidence interval, 0.0-0.9). Larger ipsilateral A1 diameters and A1/M1 diameter ratio are associated with embolic ACAS. These findings suggest that A1 diameters and M1 diameters are important in determining the path of emboli that reach the terminal internal carotid artery.